| Objective:To observe the expression of Toll-like receptor 4(TLR4) and nuclear factor-KB (NF-κB) in the myocardial tissue and discuss the protective effect and possible mechanism of simvastatin, Losartan and the combination of the two drugs, the animal model of STZ-induced diabetic rats was established, which provided experimental basis for clinical treatment.Methods:90 male Sprague-Dawley rats, weight 180-220g, were purchased from and housed in the Animal Experimental Center of Shanxi Medical University (clean environment). The rats were randomly divided into normal control group (group NC, n=18), diabetes mellitus group (group DM, n=18), Simvastatin group (group S, n=18), Losartan group (group L, n=18) and drug combination group (group C, n=18). After 12 hours of fasting, the normal control group was given citric acid-sodium citrate solution (55mg/kg) intraperitoneal injection. The remaining rats were each given the STZ (streptozotocin,55mg/kg) intraperitoneal injection. The STZ was diluted with 0.1 mol/L citric acid-sodium citrate (pH 4.40) to 1% solution. After 72 hours, if the glucose concentration of tail vein blood was greater than 16.7mmol/L and maintained for more than 3 times, then diabetic rat model had been determined to be successfully established. The groups were treated after 3 weeks. Rats in group S and group L were treated with Simvastatin(10mg·kg-1·d-1) NS MCT and Losartan(50mg·kg-1·d-1) NS MCT by gavage respectively, rats in group C were given both kinds of drugs, while the group NC and diabetes group were treated with equal volume of NS by gavage. The experimental datas, such as body weight, the blood glucose were monitored weekly. Other datas were monitored at 4th week,8th week and 12th week separately, with 6 rats at each period. Cut the tail of rats and measured the blood glucose;anesthetized the rats, took blood from vena cava, separated the serum and measured the insulin; got the hearts and dried with filter paper, measured the heart weight and figured out the heart weight index(heart weight mg/body weight g);got the cardiac muscle, partly frozen in liquid nitrogen and saved at -70℃, determined the TLR4 and NF-κB p65 mRNA of myocardial tissue by Real Time RT-PCR; partly fixed with 10% neutral formaldehyde, dehydrated with gradient alcohol,embedded in paraffin, made into paraffin section and stained with HE method, then observed the morphological changes of cardiac muscle and the area of cardiac muscle cells under the light microscope and determined the content of myocardial TLR4 and NF-κB p65 by immunohistochemistry.Results: 1.The comparison of parameters A(blood glucose and blood insulin) of each group to the corresponding period:the blood glucose of rats in groups DM, S, L and C all increased compared with those in group NC, while blood insulin all decreased, which were all of statistical significance(P<0.05).2.The comparison of parameters B(heart weight index and areas of cardiac muscle cells) of each group to the corresponding period:parameters B of group DM increased compared with those in group NC(P<0.05);parameters B of group S, L and C all decreased compared with those in group DM(P<0.05);parameters B of group C decreased compared with those in group L and group S(P<0.05). As time went on, the parameters B of group DM increased (P<0.05); the heart weight index of group S, L and C all decreased, which had no statistical significance (P>0.05); while the areas of cardiac muscle cells decreased, which were of statistical significance (P<0.05).3.The observation of myocardial morphology:the myocardial collagen fibers of group NC were thin, evenly distributed and arranged in neat rows, no pathological changes were found. The myocardial collagen fibers of group DM were coarse, partly disorder at 4th week; cardiac muscle cells were hypertrophic, loose and the myofibril fragmented at 8th week; cardiac muscle extracellular matrix increased and the fibrosis was obvious at 12th week. As time went on, the changes above of group S, L and C gradually improved, the changes of group C improved significantly over the other two groups.4.The comparison of parameters C(the expression of myocardial TLR4 and NF-κB p65) of each group to the corresponding period:parameters C of group DM increased compared with those in group NC(P<0.05);parameters C of group S,L, C decreased compared with those in group DM(P<0.05); parameters C of group C decreased compared with those in group S, L(P<0.05). As time went on, the parameters C of group DM all increased (P<0.05), while the parameters C of group S,L, C decreased, which had no statistical significance(P>0.05).5.The comparison of parameters D(the expression of myocardial TLR4 and NF-κB p65 mRNA) of each group at 12th week:parameters D of group DM increased compared with those in group NC(P<0.05); parameters D of group S, L, C decreased compared with those in group DM(P<0.05);parameters D of group C decreased compared with those in group S, L(P<0.05).6.Correlation Analysis:The expression of myocardial TLR4 of diabetes rats were positively correlated with the heart weight index(r=0.697, P<0.01)and the areas of myocardial cells (r=0.713,P<0.01).Conclusion:The TLR4 and NF-κB of myocardial tissue of diabetes rats increased and early diabetic cardiomyopathy changes emerged. Simvastatin and Losartan reduces the expression of TLR4 and NF-κB in the myocardium of diabetic rats, improved the myocardial hypertrophy and reduced the pathological damage. The combination therapy of Simvastatin and Losartan may have synergistically protective effect for diabetic cardiomyopathy.
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