Preconditioning decreases ischemia/reperfusion-induced release and activation of matrix metalloproteinase-2

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.     

Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: Vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1, of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitu-tively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila. © 1992 Wiley-Liss, Inc.     

基质金属蛋白酶与体外循环肺损伤研究进展

肺泡 毛细血管基底膜损伤是体外循环 (CPB)术后肺损伤发生和发展的主要病理过程 ,基质金属蛋白酶 (MMPs)可能通过降解细胞外基质、调节细胞因子而参与CPB所致肺损伤的发生 ,研究MMPs在CPB肺损伤中的作用机制 ,对于防治CPB术后肺损伤的发生和发展具有重要意义     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Apolipoprotein A-I attenuates renal ischemia/reperfusion injury in rats

Apolipoprotein A-I (ApoA-I), the major protein component of serum high-density lipoprotein (HDL), exhibits its anti-inflammatory activity in inflammatory responses. As renal inflammation plays an important role in ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the beneficial effect of ApoA-I on renal I/R injury in rats and the underlined mechanism. Using rats subjected to renal I/R by occlusion of bilateral renal pedicles, we found that administration of ApoA-I significantly reduced serum creatinine levels, serum TNF-α and IL-1β levels as well as tissue myeloperoxidase (MPO) activity, compared with I/R controls. Moreover, ApoA-I treatment suppresses the expression of intercellular adhesion molecules-1 (ICAM-1) and P-selectin on endothelium, thus diminishing neutrophil adherence and the subsequent tissue injury. These results showed that ApoA-I reduced I/R-induced inflammatory responses, decreased renal microscopic damage and improved renal function. It seems likely that ApoA-I protects kidney from I/R injury by inhibiting inflammatory cytokines release and neutrophil infiltration and activation.     

Inhibitory effects of EGb761 on the expression of matrix metalloproteinases (MMPs) and cartilage matrix destruction

Cytokines such as tumor necrosis factor alpha (TNF-α)-induced expression of matrix metalloproteinase (MMP) play a pivotal role in the destruction of articular cartilage in patients who are suffering from osteoarthritis (OA). Collagen type II, the basis for articular cartilage, can be degraded by MMP-1, MMP-3, and 13. EGb761, the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, has shown its anti-inflammatory capacity. This study aimed to determine a mechanism whereby EGb761 may inhibit cartilage degradation. Our results indicated that pretreatment with EGb761 abolishes MMP-1, MMP-3, and MMP-13 gene expression and protein expression induced by TNF-α in human chondrocyte monolayer. In addition, the reduction of the tissue inhibitor of metalloproteinase-1(TIMP-1) and metalloproteinase-2 gene expression induced by TNF-α was rescued by pretreatment with EGb761. Importantly, TNF-α-induced degradation of collagen type II was ameliorated by EGb761 in a dose-dependent manner. Mechanistically, our results indicated that EGb761 treatment attenuated TNF-α-induced NF-κB activation. These actions of EGb761 suggest a mechanism by which EGb761 may act to prevent cartilage breakdown in arthritis.     

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.     

Downregulation of matrix metalloproteinase-9 by melatonin during prevention of alcohol-induced liver injury in mice

Matrix metalloproteinases (MMPs) have been implicated in inflammatory and degradative processes in several diseases. The study aims to explore the mechanism of MMP-9 regulation in alcohol-induced acute liver injury and its protection by melatonin in mice. Alcohol-induced acute liver injury was induced in female Balb/C mice by ethanol administration and protection studies were carried out with a well-known antioxidant molecule, melatonin. Degree of liver injury was monitored by histological and biochemical analysis of liver tissues. Oral administration of ethanol in mouse caused significant increase in alanine amino transferase (ALT) activity in serum. Depletion of glutathione and enhancement of lipid peroxidation as well as protein oxidation was observed in liver tissues following ethanol treatment. However, melatonin exhibited potent hepatoprotective activity by inhibiting ALT activity and oxidative stress. Additionally, MMP-9 expression was increased by ethanol in a dose and time dependent manner in liver tissue and serum. Increased secretion of proMMP-9 was strongly correlated with the expression of proinflammatory cytokines e.g., tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL6. Melatonin showed hepatoprotective role by downregulation of MMP-9 and upregulation of tissue inhibitor of metalloproteases (TIMP-1) expression in liver tissue. Nuclear factor (NF)-κB, plays an important role in inducing inflammatory genes during oxidative stress, thus the role of NF-κB in ethanol-induced liver injury was investigated. Ethanol induced nuclear translocation of NF-κB and increased degradation of inhibitor of NF-κB (IκBα) in liver tissues. Moreover, ethanol-induced NF-κB translocation into nucleus was inhibited significantly by melatonin. This is the first study to elucidate the induction of MMP-9 expression by NF-κB-dependent pathway in ethanol-induced acute liver injury in mice. This study also identifies the novel role of melatonin in hepatoprotection via MMP-9 down regulation.     

Nitric oxide mediates lung injury induced by ischemia-reperfusion in rats

Nitric oxide (NO) has been reported to play a role in lung injury (LI) induced by ischemia-reperfusion (I/R). However, controversy exists as to the potential beneficial or detrimental effect of NO. In the present study, an in situ, perfused rat lung model was used to study the possible role of NO in the LI induced by I/R. The filtration coefficient (Kfc), lung weight gain (LWG), protein concentration in the bronchoalveolar lavage (PCBAL), and pulmonary arterial pressure (PAP) were measured to evaluate the degree of pulmonary hypertension and LI. I/R resulted in increased Kfc, LWG, and PCBAL. These changes were exacerbated by inhalation of NO (20-30 ppm) or 4 mM L-arginine, an NO precursor. The permeability increase and LI caused by I/R could be blocked by exposure to 5 mM N omega-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), and this protective effect of L-NAME was reversed with NO inhalation. Inhaled NO prevented the increase in PAP caused by I/R, while L-arginine had no such effect. L-NAME tended to diminish the I/R-induced elevation in PAP, but the suppression was not statistically significant when compared to the values in the I/R group. These results indicate that I/R increases Kfc and promotes alveolar edema by stimulating endogenous NO synthesis. Exogenous NO, either generated from L-arginine or delivered into the airway, is apparently also injurious to the lung following I/R.     

Association of NKT cells and granulocytes with liver injury after reperfusion of the portal vein

Reperfusion of the liver was conducted by clamping the portal vein for 30 min in mice, followed by unclamping. Unique variation in the number of lymphocytes was induced and liver injury occurred thereafter. The major expander cells in the liver were estimated to be natural killer T cells (i.e., NKT cells), whereas conventional T cells and NK cells increased only slightly or somewhat decreased in number and proportion at that time. Reflecting the expansion of NKT cells in the liver, a Th0-type of cytokine profile was detected in sera, and cytotoxic activity was enhanced in liver lymphocytes. In NKT cell-deficient mice including CD1d (-/-) mice and athymic nude mice, the magnitude of liver injury decreased up to 50% of that of control mice. It was also suspected that accumulating granulocytes which produce superoxides might be associated with liver injury after reperfusion. This might be due to stress-associated production of catecholamines. It is known that granulocytes bear surface adrenergic receptors and that they are activated by sympathetic nerve stimulation after stress. The present results therefore suggest that liver injury after reperfusion may be mainly caused by the activation of NKT cells and granulocytes, possibly by their cytotoxicity and superoxide production, respectively.     

Gender specific effect of progesterone on myocardial ischemia/reperfusion injury in rats

The study was designed to investigate the effect of progesterone and its gender based variation on myocardial ischemia/reperfusion (I/R) injury in rats. Adult Sprague Dawley rats were divided into vehicle treated reperfusion injury group male (I/R-M), female (I/R-F), ovariectomised (I/R-OVR) and progesterone treatment (I/R-M+PG, I/R-F+PG, I/R-OVR+PG) groups, respectively. I/R injury was produced by occluding the left descending coronary artery (LCA) for 1 h and followed by re-opening for 1 h. Progesterone (2 mg kg(-1) i.p.) was administered 30 min after induction of ischemia. Hemodynamic parameters (+/-dp/dt, MAP), heart rate, ST-segment elevation and occurrence of ventricular tachycardia (VT) were measured during the I/R period. The myocardial infarct area, oxidative stress markers, activities of myeloperoxidase (MPO) and creatine kinase (CK) were determined after the experiment along with the assessment of the effect on apoptotic activity by using DNA fragmentation analysis. Histological observations were carried out on heart tissue. Treatment with progesterone significantly (P<0.05) reduced infarct area, lipid peroxidation (LPO) level and activity of MPO in females (I/R-F+PG) as compared to ischemic females (I/R-F). Progesterone significantly (P<0.001, P<0.05) inhibited serum CK activity and incidences of VT in female rats. Superoxide dismutase (SOD) activity, reduced glutathione (GSH) levels were significantly elevated (P<0.05) in I/R-F+PG group. Internucleosomal DNA fragmentation was less in I/R-F+PG group when compared to I/R-F group. The ischemic male and ovariectomised (I/R-M and I/R-OVR) counterparts did not show any significant change after progesterone treatment. In conclusion, the cardioprotective effect of progesterone on myocardial I/R injury induced damage is based on gender of the animal. The protective effect could be mediated by attenuation of inflammation and its possible interaction with endogenous estrogen.     

Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Protective effects of alkaloid extract from Leonurus heterophyllus on cerebral ischemia reperfusion injury by middle cerebral ischemic injury (MCAO) in rats

The neuronal damage following cerebral ischemia is a serious risk to stroke patients. The aim of this study was to investigate the neuroprotective effects of alkaloid extract from Leonurus heterophyllus (LHAE) on cerebral ischemic injury. After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion (MCAO), some rats were intraperitoneally administered different doses of LHAE (3.6, 7.2, 14.4 mg/kg, respectively). Neurological examination was measured in all animals. Infarct volume, myeloperoxidase (MPO) activity, levels of nitrate/nitrite metabolite (NO) and apoptosis ratio of nerve fiber in brain were determined. The results showed that LHAE at 7.2 mg/kg or 14.4 mg/kg exerted significantly decreasing neurological deficit scores and reducing the infarct volume on rats with focal cerebral ischemic injury (p < 0.05). At those dose, the MPO content were significantly decreased in ischemic brain as compared with model group (p < 0.05). LHAE at 14.4 mg/kg significantly decreased the NO level compared with the model group (p < 0.05). In addition, LHAE significantly decreased the apoptosis ratio of nerve fiber compared with the model group (p < 0.05). This study suggests that LHAE may be used for treatment of ischemic stroke as a neuroprotective agent. Further studies are warranted to assess the efficacy and safety of LHAE in patients.     

The protective effect of oxytocin on renal ischemia/reperfusion injury in rats

AIM: Oxytocin was previously shown to have anti-inflammatory effects in different inflammation models. The major objective of the present study was to evaluate the protective role of oxytocin (OT) in protecting the kidney against ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Male Wistar albino rats (250-300 g) were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. OT (1 mg/kg, ip) or vehicle was administered 15 min prior to ischemia and was repeated immediately before the reperfusion period. At the end of the reperfusion period, rats were decapitated and kidney samples were taken for histological examination or determination of malondialdehyde (MDA), an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration. Creatinine and urea concentrations in blood were measured for the evaluation of renal function, while TNF-alpha and lactate dehydrogenase (LDH) levels were determined to evaluate generalized tissue damage. Formation of reactive oxygen species in renal tissue samples was monitored by chemiluminescence technique using luminol and lucigenin probes. RESULTS: The results revealed that I/R injury increased (p<0.01-0.001) serum urea, creatinine, TNF-alpha and LDH levels, as well as MDA, MPO and reactive oxygen radical levels in the renal tissue, while decreasing renal GSH content. However, alterations in these biochemical and histopathological indices due to I/R injury were attenuated by OT treatment (p<0.05-0.001). CONCLUSIONS: Since OT administration improved renal function and microscopic damage, along with the alleviation of oxidant tissue responses, it appears that oxytocin protects renal tissue against I/R-induced oxidative damage.     

Cardiac phospholipidome is altered during ischemia and reperfusion in an ex vivo rat model

Acute myocardial infarction (AMI) is the leading cause of death, morbidity, and health costs worldwide. In AMI, a sudden blockage of blood flow causes myocardial ischemia and cell death. Reperfusion after ischemia has paradoxical effects and may exacerbate the myocardial injury, a process known as ischemic reperfusion injury. In this work we evaluated the lipidome of isolated rat hearts, maintained in controlled perfusion (CT), undergoing global ischemia (ISC) or ischemia followed by reperfusion (IR). 153 polar lipid levels were significantly different between conditions. 48 features had q < 0.001 and included 8 phosphatidylcholines and 4 lysophospholipids, which were lower in ISC compared to CT, and even lower in the IR group, suggesting that IR induces more profound changes than ISC. We observed that the levels of 16 alkyl acyl phospholipids were significantly altered during ISC and IR. Overall, these data indicate that myocardial lipid remodelling and possibly damage occurs to a greater extent during reperfusion. The adaptation of cardiac lipidome during ISC and IR described is consistent with the presence of oxidative damage and may reflect the impact of AMI on the lipidome at the cellular level and provide new insights into the role of lipids in the pathophysiology of acute myocardial ischemia/reperfusion injury.     

Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.     

Effect of L-arginine and L-lysine on lysosomal hydrolases and membrane bound phosphatases in experimentally induced myocardial infarction in rats

The protective effect of L-arginine and L-lysine on lysosomal enzymes and membrane bound ATPases was examined on isoproterenol induced myocardial infarction in rats. Lysosomal enzymes play an important role in the inflammatory process. The rats given isoproterenol (150 mg kg^–1 daily) intraperitoneally for 2 days showed significant changes in the marker enzymes, lysosomal enzymes and membrane bound phosphatases. Histopathological studies also confirmed the induction of myocardial infarction in isoproterenol administered rats. Prior oral treatment with L-arginine (250 mg kg^–1 daily) and L-lysine (5 mg kg^–1 daily) for 5 days significantly prevented these alterations and restored the enzyme activities to near normal. These findings demonstrate the protective effect of L-arginine and L-lysine in combination against isoproterenol induced cardiac damage.     

Mesna (2-mercaptoethane sulfonate) prevents ischemia/reperfusion induced renal oxidative damage in rats

Reoxygenation of the ischemic tissue promotes the generation of various reactive oxygen metabolites (ROM) which are known to have deleterious effects on various cellular functions. This study was designed to determine the possible protective effect of mesna (2-Mercaptoethane Sulfonate) on renal ischemia/reperfusion (I/R) injury. Wistar albino rats were unilaterally nephrectomized, and 15 days later they were subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. Mesna (MESNA, 150 mg/kg, i.p.; an effective dose against I/R injury) or vehicle was administered twice, 15 min prior to ischemia and immediately before the reperfusion period. At the end of the reperfusion period, rats were killed by decapitation. Kidney samples were taken for histological examination or determination of the free radicals, renal malondialdehyde (MDA) and glutathione (GSH) levels, and myeloperoxidase (MPO) activity. Renal tissue collagen content, as a fibrosis marker was also determined. Creatinine and urea concentrations in blood were measured for the evaluation of renal function. The results demonstrated that renal I/R caused nephrotoxicity, as evidenced by increases in blood urea and creatinine levels, which was reversed by MESNA treatment. Increased free radical levels, as assessed by nitroblue-tetrazolium test were reduced with MESNA. Moreover, the decrease in GSH and increases in MDA levels, and MPO activity induced by I/R indicated that renal injury involves free radical formation. Treatment of rats with MESNA restored the reduced GSH levels while it decreased MDA levels as well as MPO activity. Increased collagen contents of the kidney tissues by I/R were reversed back to the control levels by MESNA treatment. Since MESNA administration reversed these oxidant responses, improved renal function and microscopic damage, it seems likely that MESNA protects kidney tissue against I/R induced oxidative damage.