| Objective:To establish models of diabetic rat and fgl2gene silencing models byLentivirus-sh-RNA of diabetic rat.To investigate the effect of cardiac function by fgl2gene silencing mideated by lentivirus shRNA in diabetic rat.;the effect of apoptosis inmyocardial cell. To explore the possibility of fgl2as target to treat diabeticcardiomyopathy and the mechanisms of molecular involved.Methods:A total of40adult male Sprague-Dawley rats, weighting180-220g, Wererandomly divided into control group(n=8)and model group (n=32). Streptozotocin(STZ) was injected by intraperitoneal in model group. diabetes rats were randomlydivided into (1) fgl2-lentvirus RNAi group (n=8）(see literature) fgl2lentvirusvector1x109IU/n, diluting by saline to1ml injected by tail vein rapidly,(2) GFP-lentvirus sh-RNAi group(n=8), GFP--lentvirus sh-RNAi1x109IU/n diluting bysaline to1ml injected rapidly by tail vein;(3) diabetes group, saline solution1mlinjected by tail vein.Natural light, average feed, free water. Tail venous blood wasextracted every4weeks and each group blood glucose, blood fat, creatinine, ureanitrogen, etc were detected. The rat heart LVEF, SF, and HR were tested byultrasonic after12weeks,then quickly open chest, TNF–alpha were detected too.Quickly taking out the heart, excess tissue was remmoved, saline rinse, weighing;longitudinally, left ventricular myocardial, myocardial tissue was fixed byformaldehyde for pathological slices. To observe the pathological morphology by HEstaining and fgl2was detected by immunohistochemical streptavidin-peroxidase,myocardial cell apoptosis was detected by TUNEL method.The expression of fgl2and apoptosis related bax, bcl-2and p38MAPK, TLR4were tested by Wersternblot.Results:(1)Diabetes group rats drinking water, eating quantity increasing, weightdecreased and color matt, urine increasing, bedding is wet. There are two dead in diabetes group, one dead in GFP lentvirus group,one dead in fgl2gene silencinggroup.On contrary the rats weight increasing, color luster, dry bedding, no dead incontrol group.(2) Compared with the4th weeks. and8th weeks. the glucouse of blooddecreased in the12th weeks. The difference of blood glucouse was significantly (P <0.05); Total cholesterol and triglyceride in8weeks and12weeks were graduallyreduced, the difference was significant (P<0.05); but the difference was no significantbetween the4and the12months in control group (P>0.05);The changes of bloodglucose and total cholesterol were unconspicuous compared with other groups forfgl2gene silencing group (P>0.05)（3) The result of cardiac function tested by ultrasonic of fgl2gene silencinggroup: LVEF, SF reduced, heart and weight ratio increased, compared with controlgroup,the difference was significantly (P <0.05), but rate of heart decreased, thedifference was no significantly (P>0.05); The difference of LVEF, SF, HR andheart and weight ratio was significantly between gene silencing group of fgl2shRNAiand both diabetes group or diabetes GFP group(P<0.05). But the difference of LVEF,SF, HR and heart and weight ratio is no significant between diabetes group anddiabetes GFP–lentivrus group (P>0.05).(4) Biopsy of myocardial by HE staining under control group rat myocardial cellarrangement is neat, it was uniform of the nucleus size, staining of cytoplasmCompare the myocardial fibers go line, diabetes group, mainly for disorderedarrangement of cardiomyocytes hypertrophy, myocardial cells, the nucleus size lessrules, has different degree of myocardial cell degeneration; Myocardial fibers opacity,and vertical and horizontal lines blurring, parts of focal necrosis, with varying degreesof coagulation necrosis; Fgl2gene silencing group of myocardial cells in the diabeticgroup improved significantly.(5) The expression of fgl2can be observed in the myocardial cells and aroundmyocardial microvascular. A lot of tan floc in myocardial microvascular around anda large number of blond in myocardial nuclei in diabetic group and GFP group.Microvascular around no floc in control group, there are very little blond in the myocardial nuclei, around the capillaries is a small amount of floc, a small amount ofmyocardial nuclei are blond in gene silencing group of Fgl2shRNAi.(6) Myocardial cell apoptosis was detected by the terminal deoxynucleotidetransferase-mediated dUTP nick end labeling (TUNEL) technique; GFP lentivrusdiabetes group and diabetes group of myocardial cell apoptosis index was30%-40%,control group of a little amount of apoptosis, apoptosis index was less than5%, RNAigene silencing group of myocardial cell apoptosis index was about15%.(7) Expression of target protein detected by western blot: compared with theother group the expression of bax/bcl-2protein was decreased in fgl2RNAi group,and bax decreased more obvious,compared with diabetes group and GFP groupthe bcl-2has no obvious difference in control group; compared with diabetes groupand diabetic GFP group the expression of p38MAPK, TLR4and fgl2protein werelowered in fgl2RNAi gene silencing group,but slightly higher than the controlgroup.Conclusion:(1)The expression of fgl2were obviously down-regulated in diabetes ratmyocardial cells in gene silencing mediated by lentivirus fgl2-shRNA.(2)Cardiac function improved in fgl2gene silencing diabetic rats mediated bylentivirus fgl2-shRNA.the mechanism may be that the expression of fgl2deceased inmyocardial microvascular or around the capillaries of diabetic rats and inhibitedapoptosis in diabetic rats myocardial cell.(3) The pathway may be related to regulating bax/bcl-2and TNF-alpha, TLR4.and etc in fgl2gene silencing diabetic rats. |
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