Inflammation Mechanism Of Myocardial Injury In Diabetic Rats And The Intervention Of Puerarin

ObjectiveDiabetic cardiomyopathy（DCM）is one of the chief diabetes mellitus （DM）complications. It has been suggested inflammation factors may be one reason fordamage which casued by DM. Thereby inhibiting inflammation factors is one of theimportant strategies to protect heart function. The aim of this research is to study theeffect of puerarin on the change of NF-κB-TNF-a-ICAM-1,COX-2signal pathway inDM rat and myocardial cells injured by high glucose. This investigation will help toexplore new therapeutic targets for diabetic cardiovascular complications and newdrug development goals. Methods1．Establish the experiment diabetes mellitus rat model50of male Sprague-Dawley （SD） rats were randomly select10as negativecontrol group，the rest rats were intraperitoneal injection of streptozotocin （STZ）with60mg/kg to establish SD diabetes mellitus model. After eight weeks, detectedmyocardial enzymes（AST、LDH、CK、CK-MB）and Masson staining to judgeif the myocardial injury and fibrosis emerged.2．Effects of puerarin on heart function in DM rat and themorphology of myocardial cells injured by high glucose⑴Then the rats in successful model were randomly divided into model group,low dose puerarin （50mg/kg）, and high dose puerarin （200mg/kg）,10rats in eachgroup. Corresponding drugs were intraperitoneally injected once a day. The animalsin negative control group and model group were given equal sodium chloride. Aftereight weeks, the effects of puerarin on the DM rats were observed byechocardiography, including left ventricular internal diameter at end-systole （LVIDs）,left ventricular internal diastolic diameter （LVIDd）, ejection fraction （EF）, fractionalshortening （FS）, and also by the detection of biochemical indices and myocardialenzymes, including fasting blood glucose （FBG）, total cholesterol （TC）, triglyceride（TG）, aspartate aminotransferase （AST）, lactate dehydrogenase （LDH）, creatinekinase （CK） and isoenzyme of creatine kinase （CK-MB）. Using the Biopec systemhemodynamics was monitored in all rats with respect to cardiac function including+dp/dtmax. Blood samples were collected from the abdominal aorta for thefollowing experiment. Left ventricular weight index （left ventricular weight/bodyweight ratio, LVWI） were calculated, and samples were collected for histologicaland ultrastructural examination.⑵The myocardial cells were randomly divided into five groups: Normalcontrol group: there is no stimulant was given to the cardiac myocytes except theDMEM culture fluid, and to cultivate for24hours; Model group: myocardial cellswere cultivated with high glucose （33mmol/L） for24hours; Puerarin intervenedgroups: different density of puerarin(10^-4mol/L、10^-5mol/L、10^-6mol/L)were added to three different groups for12hours and then used high glucose （33mmol/L） tocultivate for12hours. To decide the protective effect of puerarin on myocardial cellsmorphology, the morphology changes were examined by modified Wright’s stainingand Giemsa’s staining.3． Effects of puerarin on the NF-κB signal pathway inmyocardial of DM rat and myocardial cells injured by high glucose⑴Immunofluorescence cytochemistry was used to observe the effects ofpuerarin on NF-κB expression in myocardial cells injured by high glucose.⑵TNF-α level was determined by ELISAmethod.⑶The effects of puerarin on ICAM-1expression in DM rat heart tissues weredetermined by immunofluorescence histochemistry. The expression of ICAM-1inmyocardial cells were investigated by flow cytometry.⑷The effects of puerarin on COX-2expression in DM rat heart tissues andhigh glucose injured myocardial cells were measured by ELISA andImmunocytochemistry.Results1. Establish the myocardial injury model in diabetes mellitusAfter eight weeks，compared with the negative group, the content ofmyocardial enzymes（AST、LDH、CK、CK-MB）were increased markedly （p＜0.01） Masson staining results showed that myocardial fibrosis emerged in modelgroup. Above the phenomenon incidated the myocardial injury model wassuccessful. 2. Effects of puerarin on heart function in DM rat and themorphology of myocardial cells injured by high glucose⑴In the course of experiment the rats in negative group had no diabeticsymptoms. The rats of model group showed furs were filthy reluster and growthslowly, hyperdiuresis, polydipsia, polyphagia and emaciation markedly. Thementioned symptoms decreased in puerarin groups （50mg/kg、200mg/kg）.⑵①At the end, compared with the model group, the BW in high dosepuerarin group （200mg/kg） significantly increased （p＜0.01）, and levels of FBG、TC、TG、 AST、LDH、CK、CK-MB declined （p＜0.01）;②Echocardiographyresults indicated that compared with the model group LVIDd and LVIDs wereameliorated in the puerarin groups （50mg/kg、200mg/kg） especially in high dosegroup （200mg/kg）（p＜0.01） while FS and LVEF had significantly increased （p＜0.01, p＜0.05）, low dose group （50mg/kg） is not significant.③Compared with themodel group+dp/dtmax was significantly improved by high dose group of puerarin（200mg/kg）（p＜0.01）, low dose group （50mg/kg） is not significant （p﹥0.05）.-dp/dtmax were increased markedly in both puerarin groups （50mg/kg、200mg/kg）.④In puerarin treated groups （50mg/kg、200mg/kg） LVWI was significantlydecreased compared with the model group（p＜0.05, p＜0.01）.⑶Electron microscope results showed that myocardial mitochondrion weredegenerationed, myofibril were dissolved degeneration, myocardial fibers arrangedirregularly in the model group while the aboved phenomenon was reduced obviouslyin puerarin groups （50mg/kg、200mg/kg）.⑷The Wright’s staining and Giemsa’s staining results suggested that comparedwith the high glucose model group, puerarin groups(10^-4mol/L、10^-5mol/L、10^-6mol/L)reduced the myocardial cells morphology injury obviously.3. Effects of puerarin on the NF-κB signal pathway in DM ratand myocardial injured by high glucose⑴Compared with the model group the expression of NF-κB were decreasedalong with the increase of puerarin concentrations. ⑵The serum levels of TNF-α were remarkably declined in high dose pueraringroup （200mg/kg） compared with the model group （p＜0.01）.⑶Immunocytochemistry results showed that the content of ICAM-1weredecreased along with the increase of puerarin concentrations compared with themodel group. Flow cytometry results indicated that compared with the model group,the expression of ICAM-1in myocardial cells were decreased.⑷Compared with the model group:①the serum levels of COX-2wereremarkably declined in high dose puerarin group （200mg/kg）（p＜0.01）, while lowdose group （50mg/kg） were not significant （p﹥0.05）.②In homogenized cardiactissues the contents of COX-2were significantly declined in both puerarin groups（50mg/kg、200mg/kg）（p＜0.01）. Compared with the high glucose model group:①the cell culture supernatant levels of COX-2were significantly declined in pueraringroup(s10^-4mol/L、10^-5mol/L、10^-6mol/L)（p＜0.01）.②the content of COX-2weresignificantly declined in high (1×10^-4mol/L) and low (1×10^-6mol/L) density pueraringroups （p＜0.01） except in middle (1×10^-5mol/L) density puerarin group （p﹥0.05）.⑸Immunocytochemistry results indicated that the expression of COX-2inmodel group were higher than those in normal group. The expression of COX-2inpuerarin groups(10^-4mol/L、10^-5mol/L、10^-6mol/L) were declined especially in high(1×10^-4mol/L) and low (1×10^-6mol/L) density puerarin groups.Conclusion1. Myocardial fibrosis and myocardial enzymes increased remarkedly in STZinduced DM rats after eight weeks.2. Puerarin can improve the myocardium ultramicrostructure injury and heartfunetion in DM rat which indicated puerarin have an effect on protecting hearttissues.3. With the pretreatment of puerarin would alleviate the myocardial cells damagewhich were injured by high glucose. 4. Compared with the normal group, the expression of NF-κB、TNF-α、ICAM-1、COX-2were increased significantly in DM rat and high glucose inducedmyocardial cells.5. Puerarin exerts preventive and remedial effects on the diabetic myocardium、high glucose induced myocardial cells and significantly improve heart function inDM rat which may be related to reduce the expressions of NF-κB、TNF-α、ICAM-1、COX-2.