| 1. ObjectiveTo study and research the effect and mechanism of classical prescription in Treatise on Febrile Diseases on stubborn disease. Based on the result of our previous clinical and empirical study on Modified Taohe Chengqi Decoction, we continue to study Effects of the decoction,and its extracts by water-alcohol,n-butyl and ethyl-acetate on diabetes mellitus and its complication in myocardium and arterial aorta, and approach to its potential mechanism of action. We focus on observed its effect on cardiomyopathy in diabetic rats, including gene expression of GluT4mRNA in cadiocyte, myocardium fibrosis, calcium transport in cadiocyte, et al. Moreover, we also observed its effect on ultrastructure of myocardium and aortic arch. These will lay foundation for future study on segregate and identify its effective component.2. Content, method and result2.1 Theory and literature study.Discussed on the research direction of Treatise on Febrile Diseases subject .By analyzed progression of the subject,we deem that it should be think highly of study on rule of decoction- syndrome corresponding, decoction-desease corresponding at present, as a subject which had establish by thousands years. Study on essence of decoction-syndrome corresponding, decoction-desease corresponding and mechanism of action of decoction should be the direction of the subject.The project carry out follow the direction by studying on treatment of diabetic cardiomyopathy by classical prescription.Reviewed on the opinion and theory of pathogenisis, syndrome differentiation and treatment in diabetes mellitus by doctors in past dynasties.According to their opinion, yin deficiency and dryness-heat was the major pathogenesis , and benefiting qi and yin, moistening dryness and clearing heat were the major treatment methods in diabetesmellitus. The decoction using in the project was designed by this treatment method. Further more, we also reviewed on pathogenesis mechanism of diabetic cardiomyopathy, progression of treating diabetes mellitus by TCM, and progression of Modified Taohe Chengqi Decoction on clinical and empirical study in the past 10 years.2.2 Experimental studyDiabetic rat model was induced in streptozotocin Sprague Dawley (SD) rats by single intraperitoneal injection of 55 mg/Kg body weight of streptozotocin, divided equally into 7 groups: the decoction group, its extracted groups by water-alcohol, n-butyl alcohol, and ethyl-acetate, Diamicron treatment group ,model rats group and normal rats group. Each group were 20 rats. Rats in TCM treatment groups were lavaged of 30g/Kg body weight of crude drug , Diamicron treatment groups were lavaged of 20mg/Kg body weight of Diamicron, model rats group and nonnal rats group were were lavaged of the same doses of water, rats in normal group were fed with common forage diet, and others with high calorie forage diet. 8 weeks later, draw the materials from rats after sacrificed by dislocate its cervical vertebra.Experiment one: effects on blood glucose and blood fat in diabetic ratsBy using microamount blood-glucose meter, postprandial blood-glucose (PBG )were detected by blood drawed from cutting tail, and fasting blood glucose(FBG)> glycosylated hemoglobin(GHb)> insulin > triglyeride^ cholesterol(CHOL)> high density lipoprotein cholesterol(HDL-C) > low density lipoprotein cholesterol(LDL-C) were detected by automatic biochemistry analysator by blood drawed from oculo-orbital plexus venosus.all targets were dected twice at the beginning and the end of the experiment. Result: compared with model group, PBG decreased in all treatment group, obviously in Diamicron treatment groups and whole Decoction group (PO.05) . FBG increased in all treatment group,but amplification minimum in Diamicron groups and whole Decoction group,which was of significant difference compare with model group (PO.05) ,and n-butyl alcohol group was follow. GHb significantly increased in all diabetic rats group than normal group,the highest level was in model group but lower level was in Diamicron treatment groups, whole Decoction group,and n-butyl alcohol group. Except normal group, insulin decreased widespredly in all group, there was a significant difference when compare with pretherapy (PO.01), but negative when compare model group with other therapy group. TG increased in model group and all the therapy group, obviously in model group, Diamicron groups, ethyl-acetate group and water-alcohol group (PO.05) , the amplificationand was lower in whole Decoction group and n-butyl alcohol group. CHOL was abviously decreased in n-butyl alcohol group (PO.01) ,but increased in ethyl-acetategroup, there was no significant difference between pretherapy and posttherapy. HDL-C decreased in n-butyl alcohol group (P<0.05) ,but no significant difference between pretherapy and posttherapy. LDL-C increased in Diamicron groups but decreased in n-butyl alcohol group.Experiment two: effects on gene expression of GluT4mRNA in cadiocyte in diabetic ratsThe cardiac muscle was made into homogenate with Trizol nucleinic acid,extract total RNA according to manual ,detect gene expression of GluT4mRNA in cadiocyte in diabetic rats by reverse transcription-polymerase chain raction(RT-PCR). Result: GLUT4mRNA expression was lower in model group and all the therapy guoups, of which was lowest in model group, when compare with normal group,there was significant difference (P<0.01) ,the whole decoction group> water-alcohol,ethyl-acetate was follow as sequence.there were also significant difference when compare with normal group (PO.05) ,it was decreased in n-butanol group and Diamicron group,but the extent of decreasing was negative,and no significant difference compare with normal group.Experiment three: effects on myocardium fibrosis in diabetic ratsThe heart were segregated,and measured for the wet weight of heart-body ratio, hydroxyproline of the cardiac muscle tissue was detected by digestion method, the cardiac muscle was made into homogenate with Trizol nucleinic acid,extract total RNA according to manual, detect the mRNA expression of type I ^ and III of collagean and transforming growth factor (TGF-B) of diabetic rats' myocardium.. Result: Compare with normal group, heart-body ratio was increased in model group ^ Diamicron gruop, water-alcohol group, n-butanol group > and Ethyl acetate group(p<0.01),but there was no significant deviation between the whole Decoction group and normal group. Of hydroxyproline of the cardiac muscle tissue,there was abviously increased in model group, Diamicron group when they compare with normal group,but the whole Decoction showed significant ly decreased (p<0.01) .and other group showed no statistical significance. The mRNA expression of type I collagen decreased slightly in model rats than the normals, and decreased in all the therapy groups especially in n-butanol group. There was increased in mRNA expression of Type III collagen in model group compared to normal group. The whole decoction group > water-alcohol group were similar as normal group, that there was significant difference compared with model group(p<0.05) . There was highly significant in n-butanol group(p<0.01), mRNA expression also decreased obviously compared with Diamicron group. mRNA expression of TGF- P increased, but there was decrease in all therapy groups compare with model group and Diamicron group,of whichthe lowest n-butanol group was.Experiment four: effects on calcium transport in cadiocyte in diabetic ratsThe cardiac muscle tissues was made into homogenate,and detect Na+-K+-ATP enzyme activity and Ca2+-ATP enzyme activity of cardiac muscle cell by biochemical method, to analyze effects on calcium transport in cadiocyte in diabetic rats, result: Na+-K+-ATP enzyme activity decrease in model rats group > Diamicron treatment group, water-alcohol, n-butyl alcohol, and ethyl-acetate group.model group was of most significant,and diamicron group followed.but inversely, there was increase in the whole decoction group,and showed significant difference compared with model group or with diamicron group (p<0.01) . Ca2+-ATP enzyme activity decreased in model rats group , Diamicron treatment group, water-alcohol, n-butyl alcohol, and ethyl-acetate group.model group was of most significant,and diamicron group followed.inversely, there was obviously increase in the whole decoction group,and showed significant difference compared with model group or with diamicron group (p<0.01) .or with n-butyl alcohol group ,of with ethyl-acetate group(p<0.05). there was also significant difference when compared model group and the whole decoction group with normal group each. (p<0.05 ) . the sequence of effect on inhibiting Na+-K+-ATP enzyme activity was as follow: whole-decoction group>n-butyl alcohol group>ethyl-acetategroup>water-alcohol>Diamicron group; the sequence of effect on inhibiting Ca2+-ATP enzyme activity was as follow: whole-decoction group>ethyl-acetate group>n-butyl alcohol group>water-alcohol>Diamicron group.Experiment five: effects on ultrastructure of myocardium in diabetic rats Cardiac muscle was made into extra thin section,and observed ultrastructure of myocardium by transmission electron microscope (JEM-1200EX), conclusion: Diamicron group got the best therapy effect,and n-butanol group was similar as Diamicron group,but other goup compare with the model group, there was invariably improve ,but not as good as those in the two groupsExperiment six: effects on ultrastructure of aortic arch in diabetic rats Aortic arch was made into extra thin section,and observed ultrastructure of aortic arch by transmission electron microscope (JEM-1200EX). Conclusion: Diamicron group got the best therapy effect, ultrastructure of endothelial cell was similar as normal group,but could not find cell junction in it. internal surface of cell was rough, membrana elastica interna was integrated, smooth muscle cell and mitochondrium in tunica media were all of normal morphous, n-butanol group was similar as Diamicron group, compare other goup with the model group, there was no abvious significant difference ,but therewas invariably improve in morphous of smooth muscle cell and mitochondrium in tunica media.3.ConclusionOn blood glucose and blood fat : The whole decoction was most effective on cut down PBG, which was similar as Diamicron;the whole decoction and n-butanol extraction to prevent were most effective on prevent FBG and GHb to rise; there was no abvious effect on improving function of insulin islet function in diabetic rats, the whole decoction and n-butanol extraction were good in stopping TG rising; n-butanol extraction could low down CHOL and LDL-C.combined with all these result,we conclude that the whole decoction and n-butanol extraction of Modified Taohe Chengqi Decoction was better effect on blood glucose and blood fat in diabetic rats.On gene expression of GluT4mRNA in cadiocyte: The whole decoction of Modified Taohe Chengqi Decoction and its n-butanol , water-alcohol > ethyl-acetate extraction were good in preventing GluT4 mRNA expression of cadiocyte decrease ,and stabilize its expression, n-butanol extraction was the most effective as diamicron.On myocardium fibrosis: Modified taohe chengqi decoction and its diferent extraction maybe effective in anti-fibrosis in myocardium of diabetic rats,maintaining normal type I /III ratio,inhibiting expression of TGF- 3 1 .The n-butanol extraction showed the best effect of all the extraction.On calcium transport in cadiocyte: Modified taohe chengqi decoction and its diferent extraction maybe effective on prevent decreasing of Na+-K+-ATP enzyme activity and Ca2+-ATP enzyme activity in cadiocyte of diabetic rats, so it could prevent Ca2+ overloading invariably extent , prevent myocardium hypertrophy and patho-change by heart function failure.On ultrastructure of myocardium: Modified taohe chengqi decoction and its diferent extraction maybe effective on prevent diabetic cardiomyopathy.The n-butanol extraction showed the best effect of all the extraction.On ultrastructure of aortic arch: Modified taohe chengqi decoction and its diferent extraction maybe effective on prevent diabetic aortopathy. The n-butanol extraction showed the best effect of all the extraction.In summary, Modified taohe chengqi decoction and its diferent extraction were considerable effective on cutting down blood glucose level and blood fat level, could prevent cardiomyopathy and aortopathy in diabetic rats. The n-butanol extraction showed...
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