Ischemia-reperfusion Injury In Diabetic Rats Research Articles

Nrf2 protects against myocardial ischemia-reperfusion injury in diabetic rats by inhibiting Drp1-mediated mitochondrial fission.

Mitochondrial dysfunction and oxidative stress are considered to be two main drivers of diabetic myocardial ischemia-reperfusion injury (DM + MIRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Dynamin-related protein 1 (Drp1) play central roles in maintaining mitochondrial homeostasis and regulating oxidative stress, but the effects of the Nrf2-Drp1 pathway on DM-MIRI have not been reported. The aim of this study is to investigate the role of the Nrf2-Drp1 pathway in DM + MIRI rats. A rat model of DM + MIRI and H9c2 cardiomyocyte injury were constructed. The therapeutic effect of Nrf2 was assessed by detecting myocardial infarct size, mitochondrial structure, levels of myocardial injury markers and oxidative stress, apoptosis, and Drp1 expression. The results showed that DM + MIRI rats had increased myocardial infarct size and Drp1 expression in myocardial tissue, accompanied by increased mitochondrial fission and oxidative stress. Interestingly, Nrf2 agonist dimethyl fumarate (DMF) could significantly improve cardiac function, mitochondrial fission, and decrease oxidative stress levels and Drp1 expression after ischemia. However, these effects of DMF would be largely counteracted by the Nrf2 inhibitor ML385. Additionally, Nrf2 overexpression significantly suppressed Drp1 expression, apoptosis, and oxidative stress levels in H9c2 cells. Nrf2 attenuates myocardial ischemia-reperfusion injury in DM rats by reducing Drp1-mediated mitochondrial fission and oxidative stress.

Impacts of delta 9-tetrahydrocannabinol against myocardial ischemia/reperfusion injury in diabetic rats: Role of PTEN/PI3K/Akt signaling pathway.

Despite the current optimal therapy, patients with myocardial ischemia/reperfusion (IR) injury still experience a high mortality rate, especially when diabetes mellitus is present as a comorbidity. Investigating potential treatments aimed at improving the outcomes of myocardial IR injury in diabetic patients is necessary. Our objective was to ascertain the cardioprotective effect of delta 9-tetrahydrocannabinol (THC) against myocardial IR injury in diabetic rats and examine the role of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in mediating this effect. Diabetes was induced in male Wistar rats (8-10 weeks old, 200-250 g; n = 60) by a single injection of streptozotocin. The duration of the diabetic period was 10 weeks. During the last 4 weeks of diabetic period, rats were treated with THC (1.5 mg/kg/day; intraperitoneally), either alone or in combination with LY294002, and then underwent IR intervention. After 24 h of reperfusion, infarct size, cardiac function, lactate dehydrogenase (LDH) and cardiac-specific isoform of troponin-I (cTn-I) levels, myocardial apoptosis, oxidative stress markers, and expression of PTEN, PI3K, and Akt proteins were evaluated. THC pretreatment resulted in significant improvements in infarct size and cardiac function and decreases in LDH and cTn-I levels (P < 0.05). It also reduced myocardial apoptosis and oxidative stress, accompanied by the downregulation of PTEN expression and activation of the PI3K/Akt signaling pathway (P < 0.05). LY294002 pretreatment abolished the cardioprotective action of THC. This study revealed the cardioprotective effects of THC against IR-induced myocardial injury in diabetic rats and also suggested that the mechanism may be associated with enhanced activity of the PI3K/Akt signaling pathway through the reduction of PTEN phosphorylation.

Protective effect of Saxagliptin on diabetic rats with renal ischemia reperfusion injury by targeting oxidative stress and mitochondrial apoptosis pathway through activating Nrf-2/HO-1 signaling

Oxidative stress and apoptosis play vital role in diabetic rats suffering from renal ischemia reperfusion injury (IRI). As a dipeptidyl dipeptidase-4 (DPP-4) inhibitor, Saxagliptin(SAX)has been confirmed in the regulation of inflammation and apoptosis by targeting Nrf-2/HO-1signalling. The study was designed to explore the efficacy and potential mechanisms of SAX on inflammation and apoptosis for treating of IRI in diabetic rats. Through testing the expressions of Nrf-2, HO-1, Cleaved-Caspase9, Cleaved-Caspase3, Bax, BCL-2, Bak, Apaf-1, cytochrome C (Cytc), Cystatin C (CysC), β2-microglobulin (β2-MG), creatinine (Cr), urea nitrogen (BUN), TUNEL and pathological morphology, the effects of SAX on IRI diabetic rats have been investigatedg. The results has displayed SAX treatment significantly attenuate the cell apoptosis and pathological damage of kidney as well as lessening the expression of cleaved-caspase-9, cleaved-caspase3, Bax, cytoplasmic-Cytc, MDA, Bak, and Apaf-1 molecules, and the contents of ROS, Cr, CysC, β2-MG, and BUN. Furthermore, SAX therapy also increased the expression of Nrf-2, BCL-2, HO-1 and mitochondrial cytochrome Cytc, and enhanced the activity of SOD, CAT and GPx. Therefore, our study has indicated that SAX treatment alleviated IRI in diabetic rats by suppressing oxidative stress and mitochondrial apoptotic pathways by activating the Nrf-2/HO-1 signaling.

Bone Marrow Culture-Derived Conditioned Medium Recovers Endothelial Function of Vascular Grafts following In Vitro Ischemia/Reperfusion Injury in Diabetic Rats.

Ischemia/reperfusion injury (IRI) remains a challenge in coronary artery bypass grafting (CABG). Diabetic patients with coronary artery disease are more likely to require CABG and therefore run a high risk for cardiovascular complications. Conditioned medium (CM) from bone marrow-derived mesenchymal stem cells has been shown to have beneficial effects against IRI. We hypothesized that adding CM to physiological saline protects vascular grafts from IRI in diabetic rats. Bone-marrow derived cells were isolated from nondiabetic rat femurs/tibias, and CM was generated. As we previously reported, CM contains 23 factors involved in inflammation, oxidative stress, and apoptosis. DM was induced by streptozotocin administration. Eight weeks later, to measure vascular function, aortic rings were isolated and mounted in organ bath chambers (DM group) or stored in 4°C saline, supplemented either with a vehicle (DM-IR group) or CM (DM-IR+CM group). Although DM was associated with structural changes compared to controls, there were no functional alterations. However, compared to the DM group, in the DM-IR aortas, impaired maximum endothelium-dependent vasorelaxation in response to acetylcholine (DM 86.7 ± 0.1% vs. DM-IR 42.5 ± 2.5% vs. DM-IR+CM 61.9 ± 2.0%, p < 0.05) was improved, caspase-3, caspase-8, caspase-9, and caspase-12 immunoreactivity was decreased, and DNA strand breakage, detected by the TUNEL assay, was reduced by CM. We present the experimental finding that the preservation of vascular grafts with CM prevents endothelial dysfunction after IRI in diabetic rats. Targeting apoptosis by CM may contribute to its protective effect.

CD36 inhibition partially attenuates myocardial injury in diabetic rats with ischemic postconditioning

IntroductionTo investigate the role of CD36 (fatty acid translocation enzyme) in the myocardial ischemia reperfusion (IR) injury in diabetes with ischemic postconditioning (IPostC).Research design and methodsAdult male Sprague-Dawley rats received...

Activation of AMPK/p38/Nrf2 is involved in resveratrol alleviating myocardial ischemia-reperfusion injury in diabetic rats as an endogenous antioxidant stress feedback.

BackgroundMyocardial necrosis caused by myocardial ischemia-reperfusion (MI/R) in diabetic patients is prominently aggravated and can cause oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensing transcription factor that protects against myocardial ischemia/reperfusion injury (MIRI). However, the mechanism of action of Nrf2 in resveratrol-pretreated cardiomyocytes is complex. We assumed that adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/p38 mitogen-activated protein kinases (p38)/Nrf2 might be involved in resveratrol alleviating MIRI in diabetic rats as an endogenous protective mechanism.MethodsA total of 50 type 2 diabetes mellitus (T2DM) rat models were randomly divided into 5 groups (n=10 in each group): sham group; MI/R group; AMPK inhibitor compound C + myocardial ischemia-reperfusion (C + MI/R) group; resveratrol + myocardial ischemia-reperfusion (RSV + MI/R) group; and resveratrol + AMPK inhibitor compound C + myocardium ischemia-reperfusion group (RSV + C + MI/R) group. Rats were fed a high fat diet, and the T2DM models were established by intraperitoneal injection of 1% streptozotocin (STZ). The MIRI models were established by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion for 120 minutes. The size of myocardial infarction was measured. Serum samples were collected to measure the concentrations of creatine kinase-MB (CK-MB). The levels of lactate dehydrogenase (LDH), glutathione (GSH), and superoxide dismutase (SOD) in myocardial tissues were determined. Immunofluorescence analysis of translocase of outer mitochondrial membrane 20 (TOMM20) was performed to observe the pathological changes in myocardial tissues. The protein expressions of AMPK, p-AMPK, p38, p-p38, Nrf2, and heme oxygenase 1 (HO-1) were determined by western blotting.ResultsCompared with the sham group, the expressions of AMPK, p38, Nrf2, and HO-1 in the myocardium were significantly increased in the MI/R group. Compared with the MI/R group, the RSV + MI/R group had a significantly lower oxidative stress level, milder myocardial injury, increased expressions of AMPK, Nrf2, and HO-1, and lower expression of p38. The protein expressions of Nrf2 and HO-1 were partially inhibited in the RSV + C + MI/R group.ConclusionsResveratrol can inhibit oxidative stress and alleviate MIRI by activating the AMPK/p38/Nrf2 signaling pathway. Meanwhile, AMPK/p38/Nrf2 is also an endogenous antioxidant stress pathway that protects against stress.

Comparative evaluation of empagliflozin, canagliflozin and sitagliptin cardioprotective properties in rats with experimental type 2 diabetes mellitus

Background: Myocardial infarction (MI) is one of the leading causes of mortality in patients with type 2 diabetes mellitus (DM), therefore it is essential to give preference to a glucose-lowering drug having optimal cardioprotective properties. A comparative study of the various sodium-glucose co-transporter inhibitors representatives’ protective effects in experimental MI was not carried out within the framework of one study.Aim: To evaluate the influence of empagliflozin (EMPA) and canagliflozin (CANA), in comparison with sitagliptin (SITA), on hemodynamic parameters and myocardial damage area in rats with diabetes type 2 model in experimental MI.Materials and methods: Type 2 DM was modelled in Wistar rats by means of 4-week high-fat diet followed by nicotinamide 230 mg/kg and streptozotocin 60 mg/kg administration. 4 weeks after DM induction the following groups were made: «DM+SITA» — treatment with SITA 50 mg/kg, «DM+EMPA» — treatment with EMPA 2 mg/kg, «DM+CANA» — treatment with CANA 25 mg/kg per os once daily for 8 weeks. Animals in «DM» group remained untreated for the following 8 weeks. Rats in control group were fed with standard chow. 16 weeks after the experiment beginning transient global myocardial ischemia was modelled in all rats. Hemodynamic parameters and myocardium necrosis area were evaluated.Results: The necrosis area was larger in «DM» group, than in control one (p=0.018). Infarction size in «DM+SITA» did not differ from that in «DM» group (62.92(41.29;75.84) and 57.26(45.51;70.08)%, р=0.554). Necrosis area in «DM+EMPA» and «DM+CANA» groups was smaller than in «DM» group (37.90(20.76;54.66)%, 46.15(29.77;50.55) vs 57.26(45.51;70.08)%, р=0.008 and р=0.009, respectively). Necrosis size did not differ between «DM+EMPA» and «DM+CANA» groups (p=0.630). Ischemic contracture in «DM+CANA» group was less prominent than under the use of all other glucose-lowering drugs. We observed increase of coronary blood flow in «DM+EMPA» group, in comparison with «DM», «DM+CANA» and «DM+SITA» groups.Conclusions: SITA does not have cardioprotective effect in ischemia-reperfusion injury in diabetic rats. EMPA and CANA have similarly prominent infarct-limiting properties. EMPA is able to increase coronary blood flow, whereas cardioprotective action of CANA is associated with ischemic contracture diminishing.

The 2-(2-benzofuranyl)-2-imidazoline provides neuroprotection against focal cerebral ischemia-reperfusion injury in diabetic rats: Influence of microglia and possible mechanisms of action

Increased microglial NADPH oxidase (NOX2) production may make an important contribution to the increased incidence and severity of ischemic stroke associated with diabetes. Imidazoline receptors are closely associated with neuroprotection, but the neuroprotective effects of the selective I2-imidazoline receptor ligand 2-(2-benzofuranyl)-2-imidazoline (2BFI) in diabetes has not been established. The effect of 2BFI on microglial NOX2 production was investigated using a co-culture of neurons and microglia, and the effect on cerebral ischemia-reperfusion (IR) injury was determined in diabetic rats. Garcia neurological scores, brain infarct volumes, brain water content, TUNEL staining, blood-brain barrier, and immunofluorescent labeling for microglia were evaluated. Western blots were used to determine gp91phox and Tyr1472 expression. Anti-inflammatory cytokine (IL-10) and inflammatory cytokine secretion was determined using ELISA kits. The brain infarct volumes, TUNEL-positive neurons, expression of microglia, brain water content, blood-brain barrier structure damage, and gp91phox and Tyr1472 expression were increased, the Garcia neurological scores were significantly decreased in the IR group, and 2BFI relieved these alterations. The IL-10 concentration was increased in the IR group; 2BFI significantly improved this increase. The neuron apoptosis and necrosis rates, and production of reactive oxygen species (ROS) and inflammatory cytokines, including IL-6, IL-8, TNF-α, and 8-iso-PGF2α, were significantly increased by high glucose stimulation combined with oxygen-glucose deprivation treatment, which were inhibited by 2BFI. The 2BFI ameliorated cerebral ischemia-reperfusion injury in diabetes and decreased neuron death in an in vitro model. The mechanism underlying these findings may be related to the decreased production of inflammatory factors and reactive oxygen species from microglia.

Peripheral arterial disease: Effects of ethanolic extracts of seed kernels of mango (Mangifera indica .L) on acute hind limb ischemia-reperfusion injury in diabetic rats

Background and aimHind limb ischemia is one of the peripheral arterial diseases affecting majority of the people with atherosclerosis, diabetes and chronic cigarette smokers. Hind limb ischemic-reperfusion injury is also one of the exacerbating events in these peoples, resulting in hind limb dysfunction. The aim of this study was to identify the effects of ethanolic extracts of mangifera indica (EEMI) on reversing hind limb dysfunction in diabetic rats with acute hind limb ischemia-reperfusion injury. Experimental procedureUnilateral femoral artery ligated diabetic rats were orally fed with EEMI (0.2 and 0.4 g/kg) for 14 days. At the end of the study, plasma levels of pro-inflammatory cytokines and relevant biochemical parameters were measured. The isolated gastrocnemius muscles were used for gene expression and histopathological studies. ResultsThere was a significant reduction (p < 0.05) in the plasma levels of pro-inflammatory cytokines, nitric oxide, malondialdehyde; and the expression levels of mRNA of induced nitric oxide synthase and intercellular adhesion molecule −1; and increase in anti-inflammatory cytokine, in isolated gastrocnemius muscles of animals treated with 0.2 and 0.4 g/kg of EEMI in comparison to disease control. In addition, histopathological study of gastrocnemius muscle and hind limb function test indicated the recovery of tissue damage from ischemic reperfusion at 0.2 and 0.4 g/kg of EEMI in comparison to disease control. ConclusionWe conclude that 14-day EEMI treatment of rats with acute hind limb ischemia/reperfusion in diabetic rats recovered from ischemic/reperfusion injury by modulating (decreasing) oxidative stress and inflammation.

The Protective Role of Bmal1-Regulated Autophagy Mediated by HDAC3/SIRT1 Pathway in Myocardial Ischemia/Reperfusion Injury of Diabetic Rats

Histone deacetylase 3 (HDAC3) and silent information regulator 1 (SIRT1) are histone deacetylases that regulate important metabolic pathways and play important roles in diabetes and myocardial ischemia/reperfusion (IR) injury. In this study, we explored the protective mechanism of Bmal1-regulated autophagy mediated by the HDAC3/SIRT1 pathway in myocardial IR injury of diabetic rats. Type 1 diabetes was established by administering an intraperitoneal injection of streptozotocin. After 8 weeks, the left anterior descending coronary artery was ligated for 30 min and reperfused for 120 min to establish a myocardial IR injury model in diabetic rats. H9c2 cardiomyocytes were exposed to high glucose concentration (30 mM) and hypoxia/reoxygenation (H/R) stimulation in vitro. The myocardial infarct size and levels of serum cTn-I, CK-MB, and LDH in diabetic rats subjected to myocardial IR injury were significantly higher. Upregulated HDAC3 and downregulated SIRT1 expression were observed in diabetic and IR hearts, along with a lower Bmal1 level. Autophagy was rapidly increased in the hearts of diabetic or non-diabetic rats in the IR group compared with the sham group, but significantly attenuated in the hearts of diabetic rats compared with the hearts of non-diabetic rats after IR insult. Consistent with decreased autophagy, we observed increased HDAC3 expression and decreased SIRT1 and Bmal1 levels in the myocardial tissue of diabetic rats after IR. Inhibition of HDAC3 by the inhibitor RGFP966 and activation of SIRT1 by the agonist SRT1720 could significantly attenuate myocardial IR injury in diabetic rats by restoring Bmal1-regulated autophagy. Based on these findings, the disordered HDAC3/SIRT1 circuit (upregulated HDAC3 and downregulated SIRT1 levels) plays an important role in aggravating myocardial IR injury in diabetic rats by downregulating Bmal1-mediated autophagy. Treatments targeting HDAC3/SIRT1 to activate the autophagy may represent a novel strategy to alleviate myocardial IR injury in diabetes.

Activation of autophagy inhibits nucleotide-binding oligomerization domain-like receptor protein3 inflammasome activation and attenuates myocardial ischemia-reperfusion injury in diabetic rats.

Aims/IntroductionDiabetic hearts are more vulnerable to ischemia‐reperfusion injury (I/RI). The activation of nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome can mediate the inflammatory process, and hence might contribute to myocardial I/RI. Activation of autophagy can eliminate excess reactive oxygen species and alleviate myocardial I/RI in diabetes. The present study aimed to investigate whether the activation of autophagy can alleviate diabetic myocardial I/RI through inhibition of NLRP3 inflammasome activation.Materials and MethodsA dose of 65 mg/kg streptozotocin was given by tail vein injection to establish a type 1 diabetes model in the rats. The left anterior descending coronary artery was ligated for 30 min followed by reperfusion for 2 h to establish a myocardial I/RI model. H9C2 cardiomyocytes were exposed to high glucose (33 mmol/L) and subjected to hypoxia–reoxygenation (6 h hypoxia followed by 4 h reoxygenation).ResultsThe diabetic rats showed significant inhibition of cardiac autophagy (decreased LC3‐II/I and increased p62) that was concomitant with increased activation of NLRP3 inflammasome (increased NLRP3, apoptosis‐related spots protein cleaved caspase‐1, interleukin‐18, interleukin‐1β) and more severe myocardial I/RI (elevated creatine kinase myocardial band, lactate dehydrogenase and larger infarct size). However, administration of rapamycin, an inhibitor of the autophagy, to activate autophagy resulted in the inhibition of NLRP3 inflammasome, and finally alleviated myocardial I/RI. In vitro, high glucose inhibited autophagy, while activating NLRP3 inflammasome in H9C2 cardiomyocytes and aggravating hypoxia–reoxygenation injury, but rapamycin reversed these adverse effects of high glucose.ConclusionActivation of autophagy can suppress the formation of NLRP3 inflammasome, which in turn attenuates myocardial ischemia‐reperfusion injury in diabetic rats.

Effects of ATRA on diabetic rats with renal ischemia-reperfusion injury.

Purpose To explore the role of all-trans retinoic acid (ATRA) in renal ischemia/reperfusion injury of diabetic rats.Methods Sixty adult male rats were randomly divided into 6 groups, including sham group (S group), ischemia-reperfusion group (I/R group), ischemia-reperfusion+ATRA group (A group), diabetic group (D group), diabetic ischemia-reperfusion group (DI/R group), diabetic ischemia-reperfusion +ATRA group (DA group). The levels of creatinine (Cr), cystatin C (Cys-C) and β2-microglobulin (β2-MG) were measured. Morphology of renal tissue was observed under light microscope.Results DJ-1, Nrf2, HO-1 and caspase-3 were detected by western blot. DJ-1, Nrf2, HO-1 and caspase-3 in I/R group, D group and DI/R group was higher than that in S group. Compared with I/R group, Nrf2 and HO-1 in A group was decreased, but caspase-3 was increased. However, Nrf2 in DA group was higher than that in DI/R group, HO-1 and caspase-3 in DA group were lower than that in DI/R group. Compared with group S, Cr, Cys-C and β2-MG in I/R group, A group, D group, and DI/R group were higher. Whereas the levels of Cr, Cys-C, β2-MG and renal injury score in DA group were lower than those in DI/R group.Conclusion ATRA has a protective effect on renal ischemia-reperfusion injury in diabetic rats, maybe relating to DJ/Nrf2 pathway.

Dexmedetomidine hydrochloride up-regulates expression of hypoxia inducible factor-1α to alleviate renal ischemiareperfusion injury in diabetic rats

To verify whether dexmedetomidine hydrochloride (Dex) alleviates renal ischemia-reperfusion (IR) injury in diabetic rats by increasing the expression of hypoxia inducible factor-1α (HIF-1α). A rat model of type 2 diabetes mellitus was established by high-fat diet and streptozotocin injection. The rats were subjected to daily intragastric administration of 0.05 mg/kg digoxin for 7 consecutive days and intraperitoneal injection of Dex 2 h before renal IR injury induced by ligation of the bilateral renal arteries for 60 min followed by reperfusion for 120 min. After reperfusion, blood samples were taken for detection of serum creatinine (Scr) and urea nitrogen (BUN) levels. Western blotting was used to detect the expression of HIF-1α, cleaved caspase-3, Bcl-2, and Bax in the renal tissues; the expression of the HIF-1α, p-eNOS, and eNOS were detected using ELISA. The percentage of apoptotic glomerular cells was assessed using TUNEL assay. The levels of Scr, BUN, HIF-1α, p-eNOS, and eNOS and the percentage of apoptotic cells in both normal and diabetic rats increased significantly after renal IR injury (P &lt; 0.05). The expressions of Scr, BUN, p-eNOS, and eNOS decreased while HIF-1α expression increased significantly in Dex-treated rats with renal IR injury (P &lt; 0.05). Compared with the non-diabetic rats, the diabetic rats showed more obvious increase in the expressions of Scr, BUN, p-eNOS, and eNOS following renal IR injury. In the diabetic rats with renal IR injury, Dex treatment prior to the injury significantly lowered the expressions of Scr, BUN, p-eNOS, eNOS, cleaved caspase-3, and Bax, decreased the percentage of apoptotic cells, and increased the levels of HIF-1a and Bcl-2 (P &lt; 0.05). Digoxin treatment significantly antagonized the effects of Dex in the diabetic rats with renal IR injury by increasing the expressions of cleaved caspase-3 and Bax, promoting glomerular cell apoptosis, and decreasing renal expressions of HIF-1 and Bcl-2 (P &lt; 0.05). Dex alleviates renal IR injury in diabetic rats probably by inhibiting renal expression of HIF-1α and glomerular cell apoptosis.

Effect of sevoflurane pretreatment on myocardial ischemia-reperfusion injury in diabetic rats and the relationship with ERK1/2 signaling pathway

Effect of sevoflurane pretreatment on myocardial ischemia-reperfusion injury in diabetic rats and the relationship with ERK1/2 signaling pathway

Effect of dexmedetomidine on ischemia-reperfusion injury of liver and kidney tissues in experimental diabetes and hepatic ischemia-reperfusion injury induced rats

Background: Reperfusion following ischemia can lead to more injuries than ischemia itself especially in diabetic patients. The aim of this study was to evaluate the effect of dexmedetomidine on ischemia-reperfusion injury (IRI) in rats with have hepatic IRI and diabetes mellitus.

Effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion injury in diabetic rats: the relationship with autophagy

Objective To evaluate the effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with autophagy. Methods Clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 200-250 g, were studied.The diabetic rat model was established by intraperitoneal injection of 1% streptozotocin 60 mg/kg.Forty diabetic rats were divided into 4 groups (n=10 each) using a random number table method: sham operation group (group Sham), I/R group (group I/R), I/R plus adenosine A2A receptor agonist CGS21680 group (group CGS), and I/R plus CGS21680 plus adenosine A2A receptor antagonist ZM241385 group (group CGS+ ZM). Myocardial I/R was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120-min reperfusion.Adenosine A2A receptor agonist CGS21680 10 μg/100g was intravenously injected at 10 min before reperfusion in group CGS.CGS21680 10 ug/100g and ZM241385 0.2 mg/kg were intravenously injected sequentially at 10 min before reperfusion in group CGS+ ZM.Blood samples were obtained at the end of reperfusion for determination of concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct volume (by TTC staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3 Ⅱ, p62 and Beclin-1 (by Western blot). LC3 Ⅱ/LC3 Ⅰ ratio was calculated. Results Compared with group Sham, the serum CK-MB, LDH and cTnI concentrations and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was up-regulated, and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in group I/R (P 0.05). Compared with group CGS, the concentrations of serum CK-MB, LDH and cTnI and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in group CGS+ ZM (P<0.05). Conclusion Activating adenosine A2A receptors can mitigate myocardial I/R injury, and the mechanism may be related to enhancing autophagy in diabetic rats. Key words: Receptor, adenosine A2A; Autophagy; Diabetes mellitus; Myocardial reperfusion injury

Protective effect of insulin on myocardial ischemia-reperfusion injury in diabetic rats based on PI3K/Akt signaling pathway

Objective To study the effect of insulin on phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in diabetic rats with myocardial ischemia reperfusion injury. Methods Diabetic rats were induced by high-sugar and high-fat diet plus intraperitoneal injection of streptozotocin (40 mg/kg). They were randomly divided into diabetic sham group (group A), diabetes ischemic reperfusion group (group B), diabetes ischemic reperfusion insulin treatment group (group C) and diabetic ischemia reperfusion+ insulin Wortmaninn (PI3K inhibitors) group (group D), 10 in each group. Myocardial ischemia reperfusion model in diabetic rats: heart was exposed between the third and fourth ribs of the left chest, 2 mm from the lower edge of the left atrial ear, and 5-0 sterile suture was used to ligate the anteri-or descending coronary artery (LAD) and the great cardiac vein for 30 min, and then the perfusion was resumed for 120 min.Wortmannin (15 μg/kg) was given through femoral vein 20 min before ligation in group D, and the same amount of normal saline was given in the other 3 groups.Insulin (2 U/kg) was injected subcutaneously in group C and D 10 min before ligation, and the same amount of normal saline was injected subcutaneously in group A and group B. Plasma creatine kinase MB (CK-MB)and troponin I (cTnI) levels were measured in arterial blood after 120 min of reperfusion, and PI3K and Akt expression in myocardial tissue were detected by Western blot. Results Compared with the group A, the plasma levels of cTnI and CK-MB increased and the expressions of PI3K and phosphorylated protein kinase B (p-Akt) in myocardium decreased in the group B (P<0.05). After insulin treatment, the plasma cTnI [(0.89±0.26)μg/L], CK-MB [(9.24±3.16)μg/L] in the myocardial tissue of the group C decreased, while the expression of PI3K (0.341 8±0.03 1) and p-Akt (0.673 1±0.028) in the myocardial tissue increased (P<0.05). After insulin+ Wortmaninn administration, the plasma cTnI [(1.16±0.29)μg/L] and CK-MB [(12.57±3.01)μg/L] in the group D increased , while the expression of PI3K (0.292 7±0.036) and p-Akt (0.531 4±0.030) in the myocardial tissue decreased, with statistically significant difference (P<0.05). Conclusions Insulin can reduce serum CK-MB and cTnI levels in diabetic rats with myocardial ischemia reperfusion injury, possibly by activating PI3K/Akt signal transduction pathway, inhibiting myocardial enzyme release, and improving myocardial ischemia reperfusion injury in diabetic rats to play a protective role in myocardial cells. Key words: 1-phosphatidylinositol 3-kinase; Protein kinases; Insulin; Diabetes mellitus; Reperfusion injury; Rats

Role of class I histone deacetylase in myocardial ischemia-reperfusion injury in diabetic rats and the relationship with AMPK/mTOR signaling pathway

Objective To evaluate the role of class Ⅰ histone deacetylase (HDAC) in myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Methods SPF healthy adult male Sprague-Dawley rats, weighing 210-220 g, were used in this study.Type I diabetes mellitus was induced by single intraperitoneal injection of streptozotocin dissolved in citrate buffer 60 mg/kg, and 8 weeks later the rats with type I diabetes mellitus were used for experiment.Forty-eight diabetic rats were divided into 4 groups (n=12 each) by using a random number table method: sham operation group (group S), myocardial I/R group (group I/R), myocardial I/R plus class I HDAC inhibitor MS-275 group (group I/R+ MS) and myocardial I/R plus MS-275 plus AMPK inhibitor Compound C group (group I/R+ MS+ CC). Myocardial I/R was induced by ligation of the left anterior descending branch of the coronary artery for 45 min followed by 180 min of reperfusion in anesthetized rats.In group I/R+ MS, MS-275 10 mg/kg was intraperitoneally injected once a day for 7 consecutive days, and myocardial I/R was produced after the end of administration.AMPK inhibitor Compound C 0.5 mg/kg was intravenously injected at 30 min before ischemia in group I/R+ MS+ CC.Six rats were sacrificed at the end of reperfusion for determination of myocardial infarct size.Another 6 rats were selected at the end of reperfusion and sacrificed for determination of the level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum (by enzyme-linked immunosorbent assay), expression of AMPK, phosphorylated AMPK (p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ubiquitin-binding protein P62 (P62), microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ) and LC3Ⅱ in myocardial tissues (by Western blot). The ratios of p-AMPK/AMPK, p-mTOR/mTOR and LC3Ⅱ/Ⅰ were calculated. Results Compared with group S, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased in I/R, I/R+ MS and I/R+ M+ CC groups, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were significantly increased, p-mTOR/mTOR ratio was decreased, and P62 expression was down-regulated in group I/R+ MS (P 0.05). Compared with group I/R, the myocardial infarct size and levels of serum CK-MB and LDH were significantly decreased, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were increased, p-mTOR/mTOR ratio was decreased, and P62 expression was down-regulated in group I/R+ MS (P 0.05). Compared with group I/R+ MS, the myocardial infarct size and levels of serum CK-MB and LDH were significantly increased, the ratios of p-AMPK/AMPK and LC3Ⅱ/Ⅰ were decreased, p-mTOR/mTOR ratio was increased, and P62 expression was up-regulated in group I/R+ M+ CC (P<0.05). Conclusion Class Ⅰ HDAC is involved in myocardial I/R injury through enhancing AMPK/mTOR signaling pathway-regulated level of autophagy in diabetic rats. Key words: Histone deacetylase; Autophagy; Diabetes mellitus; Myocardial reperfusion injury; Protein-serine-threonine kinases; Receptor-interacting protein serine-threonine kinases

Antifibrotic cardioprotection of berberine via downregulating myocardial IGF-1 receptor-regulated MMP-2/MMP-9 expression in diabetic rats.

Diabetic cardiac fibrosis increases ventricular stiffness and facilitates the occurrence of diastolic dysfunction. Our previous studies have shown that berberine, a natural alkaloid, attenuates cardiac ischemia-reperfusion injury in diabetic rats. The aim of present study was to investigate the effects of long-term berberine treatment on cardiac remodeling in diabetic rats and the underlying mechanisms. Diabetic rats induced by low-dose streptozotocin injection combined with 8 wk of high-fat diet displayed significant cardiac matrix collagen deposition and dysfunction, whereas berberine administration (200 mg·kg-1·day-1, gavage 4 wk) significantly ameliorated cardiac fibrosis and dysfunction and reduced cardiac IGF-1 receptor (IGF-1R) expression in diabetic rats. Interestingly, IGF-1R expression was upregulated in cardiac fibroblasts isolated from diabetic hearts or cultured in high-glucose conditions (30 mM). High glucose treatment or IGF-1R overexpression increased matrix metalloproteinase (MMP)-2/MMP-9 expression, α-smooth muscle actin (α-SMA), and collagen type I expression in cardiac fibroblasts. In contrast, berberine treatment significantly inhibited IGF-1R expression and exerted an antifibrotic effect in high glucose-cultured cardiac fibroblasts, as manifested by decreased MMP-2/MMP-9, α-SMA, and collagen type I expression, whereas IGF-1R siRNA plus berberine treatment did not further enhance this antifibrotic effect compared with berberine treatment alone. Taken together, long-term berberine treatment ameliorates cardiac fibrosis and dysfunction by downregulating IGF-1R expression in cardiac fibroblasts and subsequently reducing MMP-2/MMP-9, α-SMA, and collagen type I expression in diabetic hearts. The findings suggest the therapeutic potential of berberine for diabetic cardiomyopathy associated with cardiac fibrosis. NEW & NOTEWORTHY Berberine downregulated IGF-1 receptor expression and matrix metalloproteinase-2/matrix metalloproteinase-9 levels in cardiac fibroblasts and thus inhibited fibroblast differentiation and collagen overproduction in diabetic hearts, suggesting a novel mechanism for antifibrotic cardioprotection of berberine in type 2 diabetes.

Role of TXNIP/NLRP3 signaling pathway in renal ischemia-reperfusion injury in diabetic rats

Objective To evaluate the role of thioredoxin-interacting protein(TXNIP)/oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)signaling pathway in renal ischemia-reperfusion(I/R)injury in diabetic rats. Methods Pathogen-free healthy male Sprague-Dawley rats, aged 8-12 weeks, weighing 200-220 g, were used in the study.Diabetes mellitus was induced by intraperitoneal injection of 1% streptozotocin 65 mg/kg and confirmed by blood glucose ≥16.7 mmol/L 3 days later.Twenty-four diabetic rats were divided into 3 groups(n=8 each)using a random number table: sham operation group(group S), renal I/R group(group I/R)and resveratrol(TXNIP inhibitor)group(group R). Resveratrol 10 mg/kg was intraperitoneally injected every day for 7 consecutive days starting from 3rd week after successful establishment of the model in group R. At 4th week after successful establishment of the model, renal I/R was produced by occlusion of bilateral renal pedicles for 25 min followed by reperfusion in anesthetized rats in group R. The animals were sacrificed at 48 h of reperfusion, and renal specimens were obtained for microscopic examination of pathologic changes and for measurement of malondialdehyde(MDA)content, superoxide dismutase(SOD)activity and superoxide anion scavenging capability(using colorimetric method), interleukin-1beta(IL-1β)and IL-18 contents(by enzyme-linked immunosorbent assay), cell apoptosis(using TUNEL)and expression of TXNIP, NLRP3 and caspase-1 in renal tissues(using Western blot). Blood samples were obtained from the left ventricle for determination of serum urea nitrogen(BUN)and creatinine(Cr)concentrations. Results Compared with group S, the serum Cr concentration and apoptosis index were significantly increased, superoxide anion scavenging capability in renal tissues was decreased, and the expression of TXNIP, NLRP3 and caspase-1 was up-regulated in I/R and R groups, and the serum BUN concentration and contents of MDA, IL-1β and IL-18 in renal tissues were increased, the SOD activity was decreased(P<0.05), and the pathological changes of renal tissues were aggravated in group I/R.Compared with group I/R, the serum BUN and Cr concentrations were significantly decreased, the contents of MDA, IL-1β and IL-18 and apoptosis index were decreased, the SOD activity and superoxide anion scavenging capability were increased, the expression of TXNIP, NLRP3 and caspase-1 was down-regulated(P<0.05), and the pathological changes of renal tissues were significantly attenuated in group R. Conclusion The pathophysiological mechanism of renal I/R injury is associated with the activation of TXNIP/NLRP3 signaling pathway in diabetic rats. Key words: Thioredoxins; NLR family, pyrin domain-containing 3 protein; Diabetes mellitus; Kindey; Reperfusion injury