| Objective It's proposed that there is a hypothesis of “phlegm and blood stasis combined each other with blocking collaterals”,and the effects of combination of Zhidang Decoction and Xiaozhanxiong Decoction(hereinafter referred to as "Didang Xianxiong Decoction")on myocardial microvascular and ECM lesions in DCM rats are studied.We set up three control groups of Huayu Tongluo,Huatan and Western medicine to explore the pathogenesis of "phlegm and blood stasis combined each other with blocking collaterals" in DCM and pathological accumulation of AGEs and the pathological changes caused by AGEs.It is proves that the intervention mechanism of " Picking evils in collaterals and Treated with Phlegm and Blood Stasis " is closely related to the accumulation and reduction of AGEs.What's more,it also promotes the clinical application of classical prescriptions in Treatise on Shanghan Lun and expanding new treatment ideas for diabetic cardiomyopathy.Methods After five days adaptive feeding,120 healthy male Sprague Dawley rats weighing 220±20g were divided into two groups randomly,one group has 110 rats for replicating diabetic(DCM)modeling,and the other group has 10 as normal groups.After 24 hours that the 110 rats were no foods to eat but given drink,the rats were throwaway injected 55mg/kg Streptozotocin(STZ)into the lower left part of the abdomen.Ultimately,a total of 90 rats eventually met the DM model criteria.Then,90 rats were divided into 6 groups again: model group,“Treatment with phlegm and stasis ”group(TY),“Disperse blood stasis and dredge collateral” group(HY),“Resolve phlegm ”group(HT),AGEs-specific protein cross-linking inhibitor group(ALT),and each group has 18 rats totally.Drug administration methods: The normal group and model group of 1.0ml/(100g·d)dose of distilled water,and three traditional Chinese medicine groups were given Didang Xianxiong decoction,Xuefu Zhuyu decoction and Xiaoxianxiong decoction with 1g/(100g·d).As for western medicine group,the positive drug ALT-711 we choose was dissolved in distilled water(dilute it when it will be used),according to 0.3mg/(100g d)decoction.All six groups were intragastrically administered once a day,and the time of gavage was set at 9 am in the morning.After 8 weeks of gavage in all rats,12 hours after fasting and forbidden water,anesthesia was Intraperitoneal injection,blood was collected from the abdominal aorta,and the heart was quickly removed.The specimens were taken and processed,and the following indicators were observed: 1 Changes of myocardial microvessels and ECM in rats:(1)HE staining of left ventricular tissue,light microscopic observation and photography;(2)Observation of myocardial cells,microvascular endothelial cells and basement membrane by transmission electron microscopy.2 Effects of the combination decoction of Didang and Xiao Xianxiong on the intervention mechanism of DCM and the accumulation of AGEs: The content of AGEs in serum and myocardial tissue was detected by ELISA method of DCM rats.(2)The expression of CTGF and its receptor LRP in myocardium was detected by Western blot and immunocytochemistry.3 Effect of the accumulation of AGEs in diabetic cardiomyopathy:(1)The expression of SR-B1?CD36 ?MSR1 proteins was detected by Western blot;(2)The expression of?SR-B1 and CD36 genes was detected by real-time PCR;Results During the experiment,a total of 22 animals died or were excluded in six groups.Compared with the model group,the generally survived situation of rats in each treatment group was improved in varying degrees.And the improvement was most obvious in TY Decoction group and positive drug ALT group.In terms of blood glucose level,although the blood glucose level of each treatment group express a downward trend,but compared with the model group,there was no significant difference(P>0.05).1(1)Ultrastructural changes of rat myocardium under transmission electron microscopy: The myocardial filaments of the normal group were arranged neatly,with no breaking,with abundant mitochondria and no degeneration,and the vascular endothelium is continuous and complete.The myofilament of myocardial cells in model group was disordered and sparse,most of them were distorted,broken,separated and partially dissolved,partial mitochondrial disorder,obviously swelling,cytoplasm vacuolar degeneration,the cristae of mitochondria were reduced and sparse,uneven thickness of capillary basement membrane,and marked nuclear membrane edema.The vascular wall was thickened and endothelial cell broke off.Compared with the model group,the myocardial ultrastructure of each treatment group was improved on different extents.The structure of cardiac myocyte in the four treatment groups was similar to that in the normal group.Myocardial cell filaments arranged neatly,nuclear membrane edema was not obvious,mitochondria in cytoplasm were abundant and less swelling.Some mitochondria had abnormal degeneration,their cristae were dense and arranged orderly,which indicated that the ultrastructure of myocardial cells was slightly damaged and the morphology was close to the normal group.2(1)Elisa method was used to determine the expression of AGEs in serum and myocardium of DCM rats:,The expression of AGEs in serum and myocardial tissue of DCM model group was up-regulated compared with the normal group,and has significant difference(P<0.01).And in comparison with the model group,the expression of AGEs was significantly decreased(P<0.01).It was found that the lowering of AGEs in ALT group was the best,followed by TY group.(2)Western blot and immunohistochemistry were used to detect the expression of CTGF and its receptor LRP in myocardial tissue: the expression of CTGF and LRP in normal group was uniformly distributed in the cytoplasm;the expression of CTGF in the cytoplasm and membrane of myocardial cells in model group was higher than that in normal group(P <0.05);compared with model group,the contents of CTGF in each treatment group has significant down(P<0.05).But there was no significant difference in LRP expression in above six groups.3(1)Detection of expression of MSR1,SR-B1 and CD36 by real-time quantitative PCR: The extents of MSR1 and CD36 in the model group significantly up in comparison with the normal group,(P<0.05)while the expression of SR-B1 was decreased(P<0.05);The expression of MSR1 and CD36 decreased to varying degrees in comparison with the model group(P<0.05),and the expression of SR-B1 increased(P<0.05);the comparison between the two groups showed that the effects of TY and ALT groups were better than group HT and group HY.(2)Western blot was used to detect the expression of MSR1,SR-B1 and CD36 in myocardium: Compared with the blank group,the expression of MSR1 and CD36 in myocardium of model group increased(P<0.05),while the expression of SR-B1 protein significant down(P<0.05);in comparison with the model group,the contents· of MSR1 and CD36 in each treatment group decreased in varying degrees,and the expression of SR-B1 increased significantly(P<0.05).The expression level of ATL group and TY group tended to be normal.Conclusion 1 The function of “Treated with Phlegm and Blood Stasis”,can interfere with the accumulation and degradation of microangiopathy,fibrotic lesions and AGEs in diabetic cardiomyopathy,which is equivalent to the effect of ALT group,indicating that this method has a good effect in preventing and treating diabetic cardiomyopathy.2 The mechanism of " Picking evils in collaterals and Treated with Phlegm and Blood Stasis " in diabetic cardiomyopathy may be related to reducing the accumulation of AGEs and accelerating its degradation.3 Didang Xiangxiong Decoction is an effective prescription for preventing and treating diabetic cardiomyopathy. |
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