| Ischemic reperfusion injury(I/R)has been an important threat in clinical Ischemic trauma and diseases.It’s meaningful to develop effective drugs on the prevention and treatment of I/R injury.Multiple mechanisms have been demonstrated to be involved in I/R injury,including Excessive production of oxygen free radicals,Neutrophil mediated inflammation and calcium overload.The clinical effect is not very ideal for the drugs used prevention and treatment of I/R injury until now.In this study,we developed a novel drug(GNN)by conjugate the nitroxide(anti-oxidative)with glycine(anti-inflammation)to prevent I/R injury.Functionally,we established two in vivo models in rats,including double hind limb I/R model and renal I/R model,to determine the protective role of GNN in local and remote tissue damages.Meanwhile,we explored the underlying mechanisms of GNN in I/R injury by using the in vitro HUVEC hypoxia/reoxygeneration model.Part one Effects of combination of ischemic post-conditioning with GNN conjugate in hind limb ischemic reperfusion injury in ratsObjective: To investigate the effects of combined treatment with ischemic post-conditioning and infusion of GNN conjugate in hind limb ischemic reperfusion animal model in vivo and to determine the protective role of GNN conjugate in remote organs during I/R injury.Methods:1 Establishment of animal model.Rats were intraperitoneally administered with sodium pentobarbital(40 mg/kg),and the body temperature was kept at 37°C using a heating pad throughout the experiments.A rubber band was used to bind the double hind limb root to cause limb ischemia.After 3 h,the tourniquet was removed to allow for reperfusion.4 hours after the reperfusion,the rats were euthanized using an i.p.injection of a lethal dose of sodium pentobarbital(100 mg/kg).The rats were randomly allocated into 5 groups:(1)Sham group: Rats subjected to the procedures as described above without I/R(n=6);(2)I/R group: Rats were exposed to hind limb ischemia for 3 h followed by reperfusion for 4 hours(n=8);(3)Ischemic post-conditioning group: four cycles of 2 min reperfusion and 2 min of re-occlusion at the onset of reperfusion combined with i.v.saline(n=8);(4)I/R injury + GNN treatment group: GNN was administrated by i.v.infusion of 10 mg/kg/min for 15 min(n=8);(5)ischemic post-conditioning + GNN treatment group: Ischemic post-conditioning(four cycles of 2 min of reperfusion and 2 min of reocclusion)combined with i.v.administration of GNN conjugate(n=8).2 Apoptosis assay for related tissues in these models.The related-tissues,including liver,kidney and muscle cells were collected and isolated with 0.25% trypsin and the apoptosis rates were determined by flow cytometry.3 Determination of related biochemical index.The serum TNF-α levels in the samples mentioned above were measured with the TNF-α ELISA kit following the manufacturer’s instruction.MDA levels were determined following Beuge’s method.Serum levels of AST,ALT,BUN and Cr were measured with an automatic biochemical analyzer.Results:1 Compared to controls,animals with the combined treatment(ischemic post-conditioning + GNN)could significantly rescue the cellular apoptotic rate in liver,kidney and muscle tissues.2 Compared with sham group(muscle: 4.02 ± 0.26;lung:3.66 ± 0.32),the rat muscle and lung W/D ratio significantly increased following I/R(muscle:6.07 ± 0.37;lung:5.73 ± 0.38)and it was attenuated by GNN conjugate alone(muscle:4.86 ± 0.38;lung:4.50 ± 0.27)or GNN combined with ischemic post-conditioning(muscle:4.45 ± 0.39;lung:4.02 ± 0.34).Meanwhile,the effect of combined treatment was more effective.3 Compared with sham group(serum: 1.67 ± 0.13 nmol/ml;lung:41.80 ± 3.20 nmol/g),the rat serum and lung MDA level significantly increased following I/R(serum:3.45 ± 0.44 nmol/ml;lung:84.50 ± 3.70 nmol/g)and it was attenuated by GNN conjugate alone(serum: 2.28 ± 0.38 nmol/ml;lung:60.30 ± 3.50 nmol/g)or GNN combined with ischemic post-conditioning(serum: 1.89 ± 0.20 nmol/ml;lung: 52.70 ± 4.90 nmol/g).Meanwhile,the effect of combined treatment was more effective.4 MPO level.The MPO level in the I/R group(19.30 ± 1.91 U/g)was significantly higher than in the sham group(8.02 ± 0.62 U/g).The induction was attenuated with GNN conjugate alone(10.41 ± 1.20 U/g)or GNN combined with ischemic post-conditioning(9.33 ± 1.31 U/g).Meanwhile,the effect of combined treatment was more effective.5 TNF-α.The TNF-α level in the I/R group(130.62 ± 5.71 pg/ml)was significantly higher than in the sham group(51.22 ± 6.90 pg/ml).The induction was attenuated with GNN conjugate alone(85.81 ± 9.12 pg/ml)or GNN combined with ischemic post-conditioning(72.20 ± 9.51 pg/ml).6 The results of BUN,Cr,AST and ALT levels showed that the function of kidney and liver was destroyed by hind limb I/R and the injury could be ameliorated by GNN conjugate.Part two Effects of GNN conjugate in renal ischemic reperfusion injury in ratsObjective: To explore the protective role of GNN conjugate in ischemic reperfusion(I/R)injury,we investigated the effects of glycine-nitronyl nitroxide(GNN)conjugate in renal I/R rat model.Methods: Establishment of an in vivo renal I/R injury model.16 male Wistar rats(4 month old,250-300 g)were fed standard food and water adlibitum,and were randomly allocated into 3 groups:(1)Control group(n=4): rats were subjected to the surgical procedures described below,except for renal ischemia/reperfusion;(2)I/R group(n=6): rats subjected to renal ischemia for 1 h followed by reperfusion for 3 h;(3)I/R + GNN conjugate treatment group(n=6): the GNN conjugate(30 mg/kg)was administered intraperitoneally to rats at 15 min before the reperfusion.The MDA levels were determined following Beuge’s method and GSH was determined based on Beutler’s method.The kidney MPO activities were measured by Hillegass’ method.The impairment of renal function was assessed by blood urea nitrogen levels,which were determined by automatic biochemistry analyzer.H&E Staninig to detect the morphology of kidney tissues under treat-ment of I/R and the GNN conjugate.Results:1 Compared to the control group rats(31.42 ± 2.80 nmol/g),the MDA levels were significantly increased in I/R group rats(72.11 ± 4.11 nmol/g).The treatment with GNN conjugate(36.51 ± 2.42 nmol/g)caused a significant inhibition of MDA production.2 The GSH level(0.81 ± 0.20 μmol/g)was decreased in the I/R group compared with the control group(1.50 ± 0.30 μmol/g).Meanwhile,the GSH levels in the GNN conjugate treatment group were close to the control group(1.41 ± 0.11 μmol/g).3 The MPO activity in I/R group(15.21 ± 0.82 U/g)was significantly elevated as compared to the control group(4.63 ± 0.73 U/g),which indicated PMN accumulation in renal tissues during I/R.The treatment with GNN conjugate(5.71 ± 0.52 U/g)decreased the MPO activity.These results imply that inflammation associated with I/Rof the kidney may be alleviated by GNN conjugate treatment.4 Compared to control group rats(14.52 ± 2.30 mg/dl),I/R group rats exhibited a significant increase in the BUN level(50.91 ± 4.21 mg/dl,P<0.001),suggesting a severe degree of glomerular dysfunction caused by renal I/R injury.After the treatment with GNN conjugate(17.51 ± 3.51 mg/dl),we observed that the level BUN was significantly reduced(P<0.001,compared to I/R group).5 Compared to the kidneys taken from control rats,the kidneys from the I/R group rats had severe cell necrosis.In contrast,the kidneys from the I/R+GNN conjugate group showed much less damage.Part three Protective role of GNN conjugate in HUVECs against hypoxia reoxygenation-induced injury and its underlying mechanismsObjective: To systematically explore the cellular and molecular mechan-isms of glycine-nitronyl nitroxide(GNN)conjugate in ischemia reperfusion(I/R),we developed an in vitro hypoxia reoxygenation(H/R)model in human umbilical vein endothelial cells(HUVECs)and determine the role of GNN conjugate in regulation of HUVECs proliferation,apoptosis,autophagy and mitochondrial morphology and function in this model.Methods:1 Isolation and culture of HUVECs.HUVECs were purified from umbilical cord of new born infant and digested with type I collagenase.The isolated cells were then plated into cell culture dish pretreated with 100 μg/ml fibronectin and identified by type VIII coagulation factor related antigens.2 Establishment of H/R model in vitro.H/R model was established when the cells were in good condition with 80% confluency.The cells ware then cultured in hypoxic medium without FBS and glucose and the culture dish was put into a sterilized hypoxic box.5% CO2 and 95% N2 was charged into the hypoxic box to decrease the concentration of oxygen(reached less than 1% after more than half an hour).Then the cells were cultured in 37°C incubator for 8 h.After that they were cultured in medium with 20% FBS with high glucose and at 37°C incubator for reoxygenation for 4 h.3 Drug treatment.Samples were divided into 4 groups,the control group(mock,wild type HUVECs),the H/R model group(T8R4),the 2 μg/ml GNN conjugate + H/R treatment group(T8R4 + D 2)and 5 μg/ml GNN conjugate + H/R treatment group(T8R4 + D 5).In the last two groups,cells were treated with 2 μg/ml or 5 μg/ml of GNN conjugate before reoxygenation were applied for the cells.4 Functional experiments.The cell proliferation was detected by MTT and apoptosis rates were determined by flow cytometry.The expression of apoptosis-related genes were examined by qPCR and western blotting.The expression and distribution of JC-1,Cytochrome c and LC3 were detected by immunofluorescence and confocal microscopy.Meanwhile,scanning electron microscope was used to examine the morphology change of mitochondria and autophagy.Results:1 After H/R treatment,the HUVECs got round and many cells floated off into the medium,however GNN conjugate could keep the HUVECs be attached to the plates with spindle-shape.Significant protective effects of GNN conjugate on cell growth in H/R-treated HUVECs were observed,which may suggest that GNN conjugate possesses a potent protective activity against H/R injury.2 GNN conjugate can rescue the proliferation rate of HUVECs with T8R4 treatment,but cannot rescue the cell cycle arrest by hypoxia/reoxygenation injury.3 H/R induced significantly apoptosis(Annexin V+)and GNN conjugate,in contrast,markedly inhibited apoptosis induced by H/R.The protective effect of GNN conjugate was also confirmed by examination of apoptosisrelated genes.qPCR and western blot analysis showed that the expressions of anti-apoptotic genes Bcl-2 and BCL-xL were decreased in H/R treated cells compared with the wild-type cells,whereas GNN treatment restored their expressions.Meanwhile,the expressions of pro-apoptotic genes Bax and BAD were up-regulated in H/R treated cells compared with the wild-type cells,whereas GNN treatment reduced their expressions.4 The morphology and function of mitochondria are changed in HUVECs under H/R treatment,including decrease of mitochondrial membrane potential,mitochondria fragmentation and expression changes of mitochondrion associated proteins.However,GNN conjugate can restore the mitochondrial morphology and function.5 H/R promotes autophagosome generation and LC3 puncta formation,while GNN conjugate decrease the number of autophagosomes and LC3 puncta.Conclusion:We systematically determined the protective role of GNN conjugate in rat renal and double lower limb I/R injury model in vivo.The results indicate that GNN conjugate not only ameliorate the in situ organ injury but also improve the function of remote organs in these models.Meanwhile,the in vitro experiments indicate that the GNN conjugate plays a significantly protective role in celluar proliferation,apoptosis,function of mitochondria and cellular autophagy in HUVECs with H/R treatment. |
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