The Protective Effect Of Lycium Barbarum Polysaccharide On Diabetic Cardiomyopathy Rats

ObjectiveHigh glucose induced H9c2 cardiomyocytes and streptozotocin（STZ）induced Sprague-Dawley（SD）rats were used as models to investigate the protective effect of Lycium barbarum polysaccharide（LBP）in diabetic cardiomyopathy.MethodsCardiomyocytes were cultured in a low-glucose DMEM medium containing10%fetal bovine serum and 1%streptomycin in a 37℃ incubator containing 5%CO₂.The cells were divided into 9 groups according to the dosing:（1）control group,（2）HG₁（50mM/l）group,（3）HG₁+LBP（30μg/ml）group,（4）HG₁+LBP（60μg/ml）group,（5）HG₁+LBP（90μg/ml）group,（6）HG₂（100mM/l）group,（7）HG₂+LBP（30μg/ml）group,（8）HG₂+LBP（60μg/ml）group,（9）HG₂+LBP（90μg/ml）group.During the experiment,cardiomyocytes were pretreated with LBP for 24 hours,then cardiomyocytes were induced with high glucose（HG）for24 hours,and adherent cells and culture medium were collected.The activity of cardiomyocytes was determined by MTT.The levels of ROS in cardiomyocytes were determined by fluorescent dye dihydroethidine（DHE）staining.In order to assess the degree of damage and antioxidant enzyme activity of cells,Malondialdehyde（MDA）concentration and glutathione（GSH）content and the activities of lactate dehydrogenase（LDH）,total antioxidant capacity（T-AOC）,superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）in cardiomyocytes were determined,Fifty rats were randomly selected from 60 adult male SD rats,and STZ（50mg/kg）was intraperitoneally injected for 5 consecutive days.Rats were considered diabetic only if they had hyperglycemia（≥16.7mmol/l）72h after one-time intra peritoneal injection of STZ.The remaining 10 SD rats served as a control group.Diabetic rats were randomly divided into three groups（n=10）:STZ group,STZ+LBP（60mg/kg/d）group and STZ+LBP（30mg/kg/d）group,The LBP group was intragastrically administered for 12 weeks.The STZ group was given the same volume of water,and the health status of the animals before and after the gavage was closely observed.At the end of the administration,rats in each group were anesthetized with 20%urethane（1g/kg）.Blood was taken from the internal iliac vein,and fasting blood glucose was measured.The body weight,heart and left ventricular weight were weighed,and the heart weight/body weight ratio（HW/BW）and left ventricular weight/body weight ratio（LVW/BW）were calculated.The mRNA of atrial natriuretic peptide（ANP）and brain natriuretic peptide（BNP）in cardiac tissue were determined by quantitative real-time PCR.The morphological changes of rat heart were observed by HE staining.The levels of tumor necrosis factor（TNF-α）and interleukin-6（IL-6）in serum were determined.Western blot analysis of intercellular endothelial nitric oxide synthase（eNOS）,inducible nitric oxide synthase（iNOS）,calpain-1,TNF-α,IL-6,adhesion molecules（ICAM）-1,vascular adhesion molecule（VCAM）-1,Smad2/3,nuclear factor-кB（NF-кB）and inhibitory proteinкB（iкB）-α,phosphorylated nuclear factor-кB（P-NF-кB）and phosphorylation inhibition proteinкB（P-iкB）-α.Immunohistochemistry was used to determine the amount of intranuclear transfer of p65（NF-кB subunit）in rat cardiomyocytes.Results（1）In vitro experiment,compared with the control group,（1）the viability of H9c2 cardiomyocytes in the HG₁ and HG₂ groups decreased to 70.63%and60.54%,respectively.（2）In the HG groups,the intracellular ROS production increased significantly.（3）In the HG groups,the vitality of LDH was significantly increased.（4）In the HG groups,the total antioxidant capacity of cardiomyocytes decreased.（5）HG treatment of cardiomyocytes increased the concentration of MDA,reduced GSH content and decreased the activity of SOD,CAT and GSH-Px.Compared with the HG group,（1）LBP treatment increased the viability of cardiomyocytes to varying degrees.（2）LBP reduced intracellular ROS production.（3）LBP reduced the leakage of LDH in cardiomyocytes.（4）LBP improved the total antioxidant capacity of cells.（5）LBP reduced MDA concentration in cardiomyocytes and increased GSH content and antioxidant enzyme activity.（2）In vivo experiment,compared with the blank group,the STZ group has the following characteristics:（1）The fasting blood glucose level of the rats was significantly increased.The heart weight/body weight and left ventricular weight/body weight ratio were increased.The cardiomyocytes in the heart tissue were disordered,and myocardial fiber bundles were loose.（2）The expression of ANP and BNP mRNA and protein increased in cardiac tissue.（3）The ROS production in myocardium increased significantly.（4）The activity of SOD and CAT and the content of GSH decreased,while the content of MDA increased.（5）The expression of eNOS protein was down-regulated in heart tissue,and the expression of iNOS protein was up-regulated.（6）The contents of IL-6 and TNF-αin serum and the protein expression of IL-6 and TNF-αin tissues increased.（7）The protein expression of ICAM-1,VCAM-1,TLR4 and smad2/3was up-regulated in tissue.（8）The protein expression of calpain-1 increased in the tissues.（9）The expression of NF-кB and P-NF-кB protein in the nucleus increased,simultaneously the expression of cytoplasmic iкB-αand P-iкB-αprotein decreased.（10）The amount of intranuclear transfer of p65 increased in cardiac tissue.Compared with the STZ group,（1）LBP decreased the fasting blood glucose levels partially,but still higher than the blank group.LBP reduced heart weight/body weight and left ventricular weight/body weight ratio.LBP improved the myocardial cells of the rat heart tissue disorder and fiber bundle looseness,（2）LBP down-regulated the mRNA and protein expression of ANP and BNP.（3）LBP decreased the content of ROS in myocardium.（4）LBP increased the activity of SOD and CAT and the content of GSH in tissues,and decreased the content of MDA.（5）LBP up-regulated the protein expression of eNOS in tissue and down-regulated iNOS expression.（6）The levels of IL-6 and TNF-αin serum and the protein expression of IL-6 and TNF-αin tissues were decreased.（7）LBP down-regulated the protein expression of ICAM-1,VCAM-1,TLR4 and smad2/3.（8）LBP reduced the protein expression of calpain-1 protein in tissues.（9）LBP decreased the protein expression of NF-кB and P-NF-кB in the nucleus of tissues,and increased the protein expression of iкB-αand P-iкB-αin the cytoplasmic.（10）LBP inhibited intranuclear transfer of p65 in cardiac tissue.Conclusion1)LBP reduces blood sugar levels in diabetic rats.2)LBP reduces the level of oxidative stress in the heart of diabetic rats.3)LBP has a protective effect on diabetic cardiomyopathy,which may be related to the reduction of oxidative stress and inflammatory infiltration,inhibition of calpain-1 activity and activation of NF-кB.