| Background:Cells in mammalian tissue can maintain normal metabolism and discharge metabolic product to vitro need sufficient oxygen and nutrient supplying.Therefore,nice circulation around cells and tissues is basic guarantee of body tissue cell metabolic activity.Due to various reasons,the amount of blood around the tissue cells reduced,which can cause the disorder of the metabolic activity,so as to cause the occurrence of ischemic injury.Basic principles of ischemic disease treatment is to restore blood perfusion to remove tissue hypoxia,thereby preventing development of ischemic injury,restore organs function,and repair the damage of organizational structure.However,in clinical trials and animal experiments,have proved that restoring the blood supplying to reestablish the circulation after the peripheral blood perfusion of the body tissue and organ decreased or interrupted for a period of time,metabolic disorders and organ damage caused by ischemia did not be alleviated,but be more serious,or even caused irreversible damage.The phenomenon of blood reperfusion causes ischemic injury exacerbate is called ischemia/reperfusion(I/R)injury.The I/R caused by shock induced by various reasons or in some important organ,such as heart,liver,kidney,brain,skeletal muscle.Skeletal muscle,as one of the components of the lower limb of the body,is one of the tissues which are very sensitive to ischemia.I/R injury in skeletal muscle occurs during limb amputation,thrombolytic therapy,limb trauma,reconstructive and transplantation surgeries,major consequences of vascular and musculoskeletal traumas.Severe ischemia reperfusion would not only cause local injury,but also oxidative stress which damages cellular macromolecules leading to toxic metabolite accumulation and systematic inflammation-related multiple organ injury.So it is very important to study the mechanism of skeletal muscle I/R injury in both clinical trials and animal experiments.At present,more and more studies have found that oxidative stress and inflammation are involved in organ damage after ischemia and reperfusion.The oxidative stress reaction is the damage of cell,molecule and organism caused by the increase of free radicals.Free radicals are the atoms of the single electron in the outer orbit which are mainly composed of reactive oxygen species(ROS)and nitrogen species reactive(RNS).Under normal circumstances,the body will continue to produce oxygen free radicals,and constantly cleared them by metabolism,to maintain a dynamic balance and lower concentration of oxygen free radicals.ROS are oxygen containing compounds or exogenous oxidants with high biological activity in the process of aerobic metabolism,whose production and the imbalance of cell antioxidant capacity can cause oxidative stress reaction.The content of ROS in normal cells and tissues is very little.It is byproducts of cell metabolism mainly found in the mitochondria,participating cell growth,differentiation and a series of physiological activities,and also can resist the invasion of microbial pathogens.Intracellular xanthine oxidase is a major source of the formation of ROS.When oxidative stress reacting,xanthine oxidase and xanthine oxidase substrate accumulation and metabolism,coactivating with lactic acid,pyruvic acid and glucose,and generating ROS.Oxygen free radicals can not only oxidate important lipids in the lipid bilayer structure to generate lipid peroxide to cause direct cellular damage,but also can cause platelet in micro vascular adhesion and aggregation,resulting in microcirculation obstacles.ROS also can further induce cell apoptosis.Many studies have indicated that the injury of organ after ischemia and reperfusion may be related to the short time exposure to high concentration of ROS.Tissue or cells will consume a large amount of ATP in the hypoxic or anoxic environment,prompting the body to produce a high concentration of ROS.ROS induce lipid peroxidation to cause ischemia and reperfusion injure.At the same time,high concentration ROS can significantly reduce activity of SOD which increased peroxidation after ischemia reperfusion,further causes organ or tissue damage.Oxidative stress has been reported to be involved in liver,skeletal muscle,brain tissue and myocardial tissue injury induced by ischemia and reperfusion.Inflammatory reaction mainly occurred in the initiation and expansion of ischemia reperfusion injury,which is an important factor to cause I/R injury.The early stage of inflammation is mainly based on neutrophil infiltration,whereas in the later stage,it is mainly the infiltration of macrophages and T lymphocytes.A large number of Lymphocytes gathered in the organization not only can block tissue microcirculation,induce the damage of cytokine,but the white blood cells in the local tissue after the aggregation of groups of respiratory effects will also induced ROS in local generation.I/R injury is characterized by necrosis of the epithelial cells and infiltration of inflammatory cells in the local necrotic tissue,and cell adhesion molecules,chemokines and cytokines are also involved in the inflammation mediated tissue damage after ischemia and reperfusion.The study showed that when the tissue ischemia and hypoxia,excess ROS produced by oxidative stress reaction will induce transcription of NF-?B.NF-?B transcription can promote a variety of inflammatory factors such as IL-1,IL-6 and TNF-?.These inflammatory cytokines can form a network of regulation and control of each other between the organirazation through autocrine,paracrine and humoral pathway to cause further tissue damage.TNF-a can induce the necrosis or apoptosis of the cells directly,which can induce the necrosis or apoptosis of the cells,and can also increase the expression of ROS indirectly,which increase the oxidative stress response after I/R.When the activity of TNF-a was inhibited,the disorder of blood brain barrier function and the necrosis of tissue after cerebral ischemia and reperfusion were obviously reduced.IL-6,as a proinflammatory cytokine,can enhance the expression of adhesion molecules.IL-1 can induce IL-8 synthesis and enhance cell adhesion molecules and integrin expression,so as to enhance the adhesion between leukocytes and endothelial cells,and form a positive feedback to further enhance the aggregation of white blood cells in the injured tissue,and to synthesize more cytokines to enhance the inflammation induced I/R injury.Lutein is a major component of green leafy vegetables,eggs and fruits,which are widely found in nature.It is an oxygenated carotenoid,highly content in serum,liver,fat and retina.The hydroxyl group present of Lutein(C40H56O2)reacts against free radicals and exerts antioxidant potential.In addition,the oxidation of Lutein products can also showed a strong clear free radicals in the body's energy,and Lutein or its product content is higher,the ability of scavenging free radicals also increase accordingly.And Lutein in food intake can significantly increase the enzyme levels of CAT?SOD?GPx and GSH.And Lutein can inhibit excessive generation of ROS in bodies.Lutein has been reported to involve in maintaining eye health and offers protection against age related macular degeneration.A number of studies suggest their potential role against oxidative stress.Lutein-induced anti-inflammation has been reported against atherosclerosis,UV-damage induced skin-inflammation,LPS inflammation in macrophages.Further protective role of Lutein against small intestine and kidney ischemic injury has been reported yet.Objective:By constructing skeletal ischemia-reperfusion model in rat,we aim to:(1)The pathological structure and leukocytes infiltration in rat with skeletal I/R injury through histology examine by lutein treatment and determine the role of Lutein protect against skeletal muscle injury.(2)By analyzing the level of MDA,protein carbonyl and sulfhydryl content,tissue ROS content as well as the expression of transcription factor Nrf-2 and determine the protective effect of lutein against oxidative stress during skeletal ischemia-reperfusion injury.(3)By measuring the total GSH content,the enzyme activity(GST,GPx,SOD,CAT),to explore the potential role of elevating the antioxidant status during skeletal I/R injury in rats.(4)Serum inflammatory cytokine(IL-6,IL-1?,TNF-?)and inflammatory transcriptional factor COX-2,NF-?B were measure to determine whether lutein treatment could significantly inhibit inflammatory responses.Methods:Fifty male Wistar rats(250-275 g)were randomly divided into 5 groups with 10 animals in each(n=10).I/R model in rats was made by using noninvasive vascular clamp with total experimental time of 6h;0.5 mg/Kg lutein was solubled in 200 ?l tea oil for intraperitoneal injection.Sham group rats were sham-operated by exposing the femoral artery and then closed the incision without I/R;Sham+Lutein(Sham+L)group rats were treated as Sham group were done and applied Lutein(0.5 mg/kg)with i.p.injection 3 h before the end of the test;I/R group rats were subjected to 4 h of femoral pedicles occlusion followed by 2 h of reperfusion and received normol saline(200 ?l)with intraperitoneal(i.p.)injection 1h before the reperfusion(i.e.,3 h before the end of the experiment);I/R +Oil(I/R +O)group rats were subjected to 4 h of femoral arteries occlusion followed by 2 h of reperfusion and applied tea oil(200 ?l)with intraperitoneal(i.p.)injection 1 h before the reperfusion;I/R +Lutein(I/R +L)group rats were subjected to 4 h of femoral arteries occlusion followed by 2 h of reperfusion and applied lutein(200 ?1)with intraperitoneal(i.p.)injection 1 h before the reperfusion.After completing the operation,the skeletal tissues were preserved for pathological structure analysis,oxidative stress parameters(MDA,ROS,protein carbonyls,protein sulfhydryls)and antioxidant parameters(GST,GPx,CAT and SOD activity assay,tissue GSH assay,expressions of Nrf-2 levels).The inflammatory mechanism was determined through expressions of NF-?B and COX-2 levels.Pro-inflammatory cytokines(IL-6,TNF-? and IL-1?)were determined by ELISA.Results:(1)Compared with Sham group,disordered muscle fibres,angiotelectasis,cell swelling as well as infiltration of leukocytes was determined in I/R group and I/R+O group.Compared with I/R group,Sham+L group could significantly reduce local leukocytes accumulation and maintain the intact structure of rat skeletal muscle.There was no significant difference between Sham group and Sham+L group,so was I/R group and I/R+O.(2)Compared with Sham group,ROS and MDA content significantly increased(P<0.001),protein carbonyl and sulfhydryl also increased obviously(P<0.001)in I/R group and I/R+O group.Sham+L group treated with Lutein showed decline in the ROS and MDA content(P<0.001),protein carbonyl and sulfhydryl level(P<0.001),compared with I/R group.There was no significant difference between Sham group and Sham+L group,so was I/R group and I/R+O.(3)Compared with Sham group,total GSH content,antioxidant enzymes(SOD,CAT,GST,GPx)activity significantly decreased(P<0.001,P<0.01,P<0.01,P<0.001,P<0.01 respectively),the expression of transcriptional factor Nrf-2 was significantly reduced(P<0.001)in I/R group and I/R+O group.Compared with I/R group,Sham+L group showed enhanced total GSH content,antioxidant enzymes(SOD,CAT,GST,GPx)activity(P<0.001,P<0.01,P<0.01,P<0.001,P<0.01 respectively),and the expression of transcriptional factor Nrf-2(P<0.001).There was no significant difference between Sham group and Sham+L group,so was I/R group and I/R+O.(4)Compared with group Sham group,in I/R group and I/R+O group,the concentration of IL-6,IL-1? and TNF-a in blood increased significantly(P<0.001),the expression of NF-?B and COX-2 was also increased obviously(P<0.001).In Sham+L group the concentration of IL-6,IL-1? and TNF-a were significantly lower(P<0.01,P<0.01,P<0.001 respectively),the expression of NF-?B and COX-2 decreased obviously(P<0.001)compared with I/R group.There was no significant difference between Sham group and Sham+L group,so was I/R group and I/R+O.Conclusions:The mechanism of Lutein alleviates I/R injury in rat skeletal muscle includes:(1)Lutein protects against skeletal muscle I/R injury,reduces local leukocytes accumulation and maintain the intact structure of rat skeletal muscle.(2)Lutein protects against skeletal I/R injury through inhibiting oxidative stress,reduces ROS,MDA,protein carbonyl and sulfhydryl content.(3)Lutein protects against skeletal I/R injury through enhancing antioxidant status,enhances total GSH content,antioxidant enzymes(SOD,CAT,GST,GPx)activity and the expression of transcriptional factor Nrf-2.(4)Lutein protects against skeletal I/R injury through inhibiting inflammatory responses,reduces the concentration of IL-6,IL-1?,TNF-a,and the expression of NF-?B and COX-2. |
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