| 1. objective:1. To investigate the Risk factors of diabetic macrvascular complication in type 2 diabetic patients;2. To observe the effects and mechanismgaster of vasculopathy and metabolic disorder (glucose, lipoids andfree radicle) on diabetic Gastroparesis)rats's delayed gastric emptying;3. To observe the effects and mechanismgaster of metabolic disorder on gastric vasculopathy;4. To observe anomalism haemorheology and inflammatory factors on DGP rat vasculopathy;5. To research the effect of vasculopathy on the structure and function impairment of DGP rat gaster and pancreatic island;6. gut hormone and neurotransmitter parasecretion and unit of gastric motility(UGM) impairment.induced by ischemia and hypoxia.2. method2.1.clinical experiment146 T2DM patients were randomly accepted from January 2002 to -March 2005 consistent with the accepted standard, include male 82, female 64;age 32-70 years ol;course of diabetes 1-78 month;non-macroangiopathy 55, macroangiopathy 91. To observe, measure and record the sequentiae items: (1) age(years),sex(0- female,1-male);(2) course of diabetes (month);(3) educational level (1-primary and secondary school, 2- senior high school and senior training school, 3- university and above);(4)diabetic complication (nephropathy, cataract and/or retinopathy and other ophthalmopathy, neuropathy, 0-no, 1-yes));(6) physical work intensity (0-light, 1-heavy);(7)mental labour intensity (0-light, 1-heavy);(8) sparetime spend on training (l-<30min per day, 2-30-60min per day, 3->60minper day);(9) patient history (diabetic family history,hypertension history or hepatopathy;0-no, 1-yes);(10) measured body height and weight, calculate habitus coefficient =height (cm)/ weight (kg);(11)to measure blood pressure, and record contractive pressure and diastolic pressure (mmHg);(12) to test second lead of electrocardiogram (0-normal, 1-abnormality), simultaneous recording average heart rate (beats per minute);(13)High Definition color transonogram was used to observe arteria carotis, arteria brachialis, arteria iliaca and arteria cruralis macroangiopathy, if one or more than one of the four great vessels has pathological changes, then this patient was accepted as DMAPpatient, if none of the four great vessels has pathological changes,-then this patient was accepted as non-DMAP patient (0 -non-DMAP- patient, 1- DMAP patient),, (14)to check content of fasting blood glucose (mmol/L), glycosylated hemoglobin (%), cholesterol total (TC), triglyeride (TG), high density lipoprotein (HDL-C) and low density lipoprotein-C (LDL-C) (mmol/L) <> two-state variable Logistic conditional forward stepwise regression was used to obtain the risk factors of DMAP. 2.2. animal experiment131 SD male rats were randomly divided into two groups: 10 rats were divided into the control group, 121 rats into DM model group, after 12h-fasting, all animals were measured fasting blood glucose(FBG) in the next morming, to reject the rats with blood sugar deviated far from the normal range, and the accepted rats were weighed,then peritoneal injected streptozotocin(STZ) citric acid-citrate sodium buffer solution, 60mg per kilogram body weight only once, to reproduce DM rat animal model.;the animals in contro lgroup were injected isastericcitric acid-citrate sodium buffer solution 0.1mol/L(pH4.5)o Im 7th dsys after STZ were given, all of the animals were tested FBG, FBG=16.7 mmol/L were accepted as DM rats, else were discarded,. All the DM rats were divided randomly into model group(about 20 rats)N aglumin group (about 20 rats)% lumbrukinase group (about 30 rats)and Tang-Wei-Kang(TWK) group(about 30 rats). In the successive 30 weeks from the 10th day, animals in TWK group were given TMK apozem (Including dangshenlOg, atractylodes macrocephala (parched) lOg, Tuckahoe 12g, prepared radix glycyrrhizae 6g, massa medicata fermentata lOg, prepared rhizoma pinelliae with alumen radix glyrrhigae calcaren 12g, bupleuri,radix 8g, aurantii fructus immaturus lOg, Salvia miltiorrhiza 12g, chuanxiong lOg et al.)p.o.;animals im aglumin groups were given water-soluted aglumin p.o.;animals in lumbrukinase group were given lumbrukinase i,p.;three drugs dose are equel to 2 times Adult's clinic commonly used dose;animals in model and control group were given isometry distilled water p.o.jall of the groups animals were given drugs or distilled water 6 times per week,and on Sunday no drugs and distilled water were given just nomal food and drink. To observe and record animsls' common status (including to drink and eat, excrete, and secrete;automatic action, mental state, pelage, cataract and even death et al.)o To weigh animals' body weight every three weeks, recount drugs dose according to the new animals' body weight.In 17th and 30th week after STZ were given,FBG was measured once. In 30th weeks,after 20 hours fasting diet, all animal were given 5ml lmg/ml phenolsulfonphthalein volumetric solution p.o.,30 minutes later, anesthetize animals with aether,then slivered cavitas thoracis and obtain blood from heart instantly.A half of animals of every group were randomly seledtde to get blood, one is 2ml ,added heparin for anticoagulation;another added no heparin, and to get no less than 1.5ml blood serum after blood clotting, -20 "C preserved to measured insulin and C-peptide in blood serum through radio-immunity method, blood fat(TC^TG^LDL^HDL, enzymic method), SOD activity, MDA content, and circulating endothelial cell(CEC);the left animals in every groups were reciped 4ml blood, after citrate sodium was used for anticoagulation, to measure haemorheology on the condition of 37 °C: whole blood viscosity, plasma viscosity, whole blood reduced viscosity, blood sedimentationrate(Wintrobe tube), hematokrit(Westergren method), fibrinogen content(ammonium sulfate turbidimetric method),whole Blood viscosity was classified ioto high shear rate 150s"1, middle shear rate 60s"1, low shear rate. 10s"1.After blood was get form heart , all animals were killed and cut the belly open, ligated cardia and pylorus of stomach, isolated and took out the whole gaster, collected Gastric contents, 722 type spectrophotometer,waa used to measure optical density value,and calculated the rate of gastric emptying. Diveded the whole tissue of sinus ventriculi into three parts, fixedby three different kinds of fixation fluid: neutral formalin, paraformaldehyde and glutaral;glutaraldehyde,then the whole tissue of sinus ventriculi were embed in paraffin block,and to obverse nest contents: ?HE,Mallory,Masson and toluidine blue dyeing to observe pathological changes of gastric wall, vascellum, and smooth muscle;myenteric plexus nerve cell;tigroid body in living myenteric plexus nerve cell. ?T immunohistochemical stain method to observed TNF-a, IL-1B, CD34, SP, VIP, 5-HT, NOS, C-kit, Bcl-2 and Fas expressed in tissue of gastric wall.to weigh pathological changes of inflammatory factor, gatric micrangium, gut hormones(GHs) and, neurotransmitters,ICC, and smooth muscle cell apoptosis gene expression,TUNEL method to obverse smooth muscle cell gene expression.at the same time. (Dglutaraldehyde fixed gaster tissue were used to be made transmission electron microscope section? to observe ultramicrostructure changes of the tissue between longitudinal muscle and sphincter.@to obtain tissue of cauda pancreatic,HE and aldehyde-fuchsin-bright green-orange G dyeing, and survey pathological changes of islands of pancreas.3. result3.1. clinical research142 effective cases selected from 146 DM patients were conducted to experiment, 89 cases with DMAP and 53 cases without DMAP were final diagnosed by B type transonogram, the same result was anticipated by regression equation, the anticipation rate was 100% which hinted that the equations had better fitting after 16 times iteration.The affection of DMAP was related to DM course, age ,culture level, practice time, oculopathy complication of DM, case history(high blood pressure , DM family history, hyperlipemia), body type coefficient, electrocardiographic abnormality, contractive pressure, heart rate, blood-fasting sugar, urinary albumin, TG and TC, etc. Among these factors, DM course, age , oculopathy complication of DM, case history(high blood pressure,DM family history, hyperlipemia), body type coefficient, electrocardiographic abnormality, contractive pressure, heart rate, blood-fasting sugar, urinary albumin, TG and TC were considered as independent risk factor of DMAP, however, culture level and practice time might be protection risk of it. These risk factors could be graded according to the importance to DMAP, blood-fasting sugar might be the most important risk factor, next were contractive pressure and hyperlipemia, the following risk factors were age , high blood pressure ,DM family history ,electrocardiographic abnormality, heart rate, urinary albumin, TC, culture level, practice time, oculopathy complication of DM, body type coefficient, TG, while DM course had little influence on DMAP. After the regression equation founded, the variance was rejected one by one and therest maintained, the logistic negative square diversity value of each rejected variance likelihood ratio change significant difference equations. Statistics analysis prove that chnges of -21og likelihood ratio had significantly difference, when vsriances in the equations quited (P<0.01)oGender, physical labor intensity, DM neuropathy, DM nephrosis, diastolic blood pressure, glycosylated hemoglobin, low-density lipoprotein and high-density lipoproteins might not be independent risk factors of DMAP, because these factors could not be adopted to the equations, Statistics analysis prove that all of these deleted vsriances had no significantly difference, when them quited (P<0.01)o3.2. animal experiment3.2.1. general introductionDuring the experiment, normal group rats obviously increased the food and drink and urinary volume, and their weight gradually raised as well, there were no abnormalities in mental status, autonomic activities, secretion, all the rats with smooth and bright fur had neither cataracta nor death.However, model group rats slowly increased the food and drink and urinary volume, as well as the weight of themselves. With loosened and lusterless fur, the symptoms of cataract, psycho-tired, autonomic activities decreasing in the rats appeared and aggravated gradually, some of them were dead. All the symptoms in TangWeiKang group and lumbrukinase group were lower than that in model group, and the opposite result was found in ethamsylate group. 3.2.2.The death of rats and body weight variationThe quantity of dead rats in lumbrukinase group , TangWeiKang group and ethamsylate group growed downwards and no dead rat in normal group, The quantity of dead rats in the early stage was more than that in the late stage . The increasing trend of rats weight in normal group showed type "S", in Chinese medince and ethamsylate group, the rats weight went down initially then increased slightly. The trend of rats weight in model group and lumbrukinase group descended in the beginning ,then ascended and dropped down in the end.There were no significant difference in the weight of each group before the model were made, at 3,6,9,12,15,18,21,24,27 and 30 weeks after the model were made, the rats weight in all model groups( model group, ethamsylate group, lumbrukinase group and TangWeiKang group) were significant lower than that in normal group, no significant difference was found in model groups. 3.23. serum glucose variation of ratsAt the 7th day after the model were made, 111 model rats could be used for experiment, the achievement ratio of model duplicating was 91.74%, serum glucose varied from 16.72 to 24.1mmol/L(averagel9.0±1.65 mmol/L). serum glucose in normal group fluctuated within normal' limits, while serum glucose in model group, ethamsylate group and lumbrukinase group step up gradually, serum glucose in TangWeiKang group decreased obviously after temporarily increased, at the 30th week after the model were made, the serum glucose in model group, ethamsylate group and lumbrukinase group were significant higher than that in normal group. While the serum glucose in TangWeiKang group was significant lower than that in model group and ethamsylategroup and no significant difference between TangWeiKang group and lumbrukinase group. 3.2.4. the pancreatic island pathological changes of rats30 weeks later, By HE dyeing, the result showed that no changes in normal group rats, the pancreatic island in model group were fewer and smaller and cell disorderly arrangement, capillary absence, cell infiltration appeared. The pathological changes in ethamsylate group were more severe than that in model group, but the adverse trend were found in lumbrukinase group and TangWeiKang group.The kytoplasm a cell, kytoplasm of 0 cell, gland alveolus epidermis of pancreas in normal group showed respectively orange yellow, prunosus, yellow brown by aldehyde-fuchsin G dyeing. ￡ cell located mainly in the center of pancreatic island, but P cell in model group were fewer and smaller. The pathological changes mentioned above in the group from severe to lightly were ethamsylate group, model group, lumbrukinase group, Chinese medicine group. The average optical, average gray scale of & cell, blood vessel density in model group were significant lower than that in normal group, while the four targets in TangWeiKang group were significant higher than that in ethamsylate group, model group, lumbrukinase group. 3.2.5.DGP rat gaster fluid Emptying RateIn 30? week after DM rat model was duplicateed, all of the DGP rats' ( model, Lumbrukinase, aglumin and TWK group) gaster fluid Emptying Rates were significant decreased, compared with control group(P<0.01);rats' gaster fluid Emptying Rates of TWK group was sigmificantly higher than model , Lumbrukinase.and aglumin three groups (P<0.01)o 3.2.6. measurement indexies mensurationIn 30 week after DM rat model was duplicateed, contents of blood-serum insulin, C-peptide, TC, TG, and LDL;SOD activity, gaster VEC topography measurement data (average optical, average gray-scale, average distribution density);dyeing areae percentage of myenteric nerve plexus nerve cell, tigroid body of nerve cell,SP, VIP, 5-HT, c-kit, and smooth muscle bcl-2;all of these items mentioned above in model group were significantly lower than control group(P<0.05). Contents of HDL-C, MDA and fibrinogen;whole blood viscosity (high shear rate, middle shear rate and low shear rate);plasma viscosity;hematocrit;indeies of Erythrocyte aggregation and deformation;areae percentage of TNF- a ,CEC, NOS and Fas;apoptotic index of smooth muscle cell, these items of model group were sigmificantly higher than control group (P<0.05), but erythrosedimentation and 5-HT areae percentage of model group were not significantly different from lumbrukinase group (P>0.05).Blood serum insulin, C- peptide and HDL-C contents;SOD activity;Erythrocyte aggregation indes;gaster VEC topography measurement data (average optical, average gray-scale, average distribution density);dyeing areae percentage of myenteric nerve plexus nerve cell, tigroid body of nerve cell, SP, VIP, 5-HT, c-kit, and smooth muscle bcl-2;all these items of TWK group were. Significantly higher than control group (P<0.05). TC, TG, LDL, MDA and fibrinogen contents> whole blood viscosity (high shear rate, middle shear rate and low shear rate);plasma viscosityjhematocrit;Erythrocyte deformation index;areae percentage of TNF- a , CEC;and smooth muscle cell apoptotic index, items of TWK group were significantlylowerer than control group (P<0.05);red blood cell deformation, 5-HT,nNOS and Fas were not significantly different from model group (P>0.05);HDL-C, hematocrit, 5-HT and nNOS were not significantly different from dicynone group (P>0.05).Blood serum insulin contents;SOD activity;whole blood reduced viscosity;Erythrocyte aggregation indes;gaster VEC topography measurement data (average optical, average gray-scale, average distribution density);dyeing areae percentage of myenteric nerve plexus nerve cell, c-kit, and smooth muscle bcl-2;all these items of TWK group were significantly higher than lumbrukinase group (P<0.05). C- peptide ,TG, HDL contents, whole blood middle and low shear rate viscosityes;hematocrit and Erythrocyte aggregation index;dyeing areae percentage of myenteric nerve plexus nerve cell, tigroid body of nerve cell, SP, VIP, 5-HT, nNOS, and smooth muscle cell apoptotic index and Fas;were not significantly different from lumbrukinase group (P>0.05). 3.2.7 the pathology and infrastructure changes of stomach blood vesselThrough light microscope, we found that the stomach capillaries of normal group were no abnormalities. Some stomach capillaries of model group were dilation and several capillaries were stenosis even obstruction. Meanwhile, less vascular smooth muscle, obvious tubal wall fibrosis, endodermis absence, basilemma thickening and blood cell adhesion also appeared in some model group capillaries. The pathological changes mentioned above in ethamsylate group were most severe because of the thrombogenesis. Without thrombogenesis, The pathological changes in TangWeiKang group and lumbrukinase group were slightly. The same result could be found through electron microscope, in addition, we detected some other phenomenon in model group such as the pathological changes of neuron and tigroid body in sinus ventriculi myenteric nerve plexusThrough Masson dying, the cell nucleus of neuron were black, the kytoplasm was yellow or brown . Toluidine blue could dyed tigroid body blue black, however, unreactive neuron were slightly dyed or could not be dyed because of less tigroid body.The neurons of myenteric nerve plexus were cluster and bead-like hypodispersion, it had big cyton, trachychromatic nucleus, abundant kytoplasm. The ganglions were consisted of several neurons with clear demarcation. The neuron cell clusters in model group were less or absence, which made the distance of adjacent clusters long and made the cluster volume small.. The neurons in the clusters with ambiguity boundary had vacuolar degeneration, less tigroid body and understain. Using electron microscope, clear nucleus, abundant cell organ and lots of chondrosome and rough endoplasmic reticulum were displayed in sinus ventriculi neurons of normal group rats , many synaptic vesicles were located in varicosity and similar electron density was found in nerve terminal.Model group rats had less sinus ventriculi myenteric nerve plexus and neurons, the details related to neurons showed as follows: nucleus and kytoplasm dissolution, chondriosome engorgement, rough endoplasmic reticulum distension. Some neurite and dendrite terminals appeared engorgement or dissolution, some of them vacuolated. There were obvious less synaptic vesicle in varicosity. The above metioned pathological changes improved in lumbrukinase group and Chinese medicine group, The pathological changes in ethamsylate group were worse than those in modelgroup.3.27. pathological changes of sinus ventriculi smooth muscleHE and Mallory dyeing shown that animals in model group had pathological changes as followed: sphincter depauperated, spatium intermusculare broaden, thickness of smooth muscle depauperatd, SMC arrangement disorder, muscle fibrosis, lightly dyeing and vacuole;vacuolus degeneration, sphincter fibrosis was lighter than longitudinal muscle. Many plasmolysis vacuoles appeared in SMC, mitochondria engorged, vacuole degeneration and dissolved. All of these pathological changes were shown worst and biggest scope in animals of aglumin group,while animals of lumbrukinase were improved,and TWK group improved most. 4.2 Animal Exprement Research(1)DM rat model is made by peritoneal injection with 60mg/kg STZ one time, and after 30 days, it is very evident of the delayed gastric emptying time of phenolsulfonphthalein, gut hormone derangement and damaged UGM, which suggest that DGP rat model has been duplicated successfully. (2) The evident metabolism derangements of sugar, grease and free radicles may be the original factors of capillary diseases which happen on the stomach of DGP rat.(3)Evident hemorheology changes in DGP rat can induce tissues and blood vessel to ischemic and hypoxia, at last lead to capillary damages.(4) The overexpression of inflammatory factor TNF-a may result in capillary damages.(5) There are the following manifestations in the stomach of GDP rat: incrassate basement membrane of medium vessels and small vessels, fibrosis of vascular smooth muscle, reduction and stenosis of capillary, microthrombus and ultramicro damages of VEC. All those may lead to the UGM damges of stomach..(6) SP and 5-HT express too less, but VIP does too more in the stomach of GDP rat. All those compose the humoral and neural factor of extreme hypofunction of the stomach.(7) Active neurocytes become less and happen vacuolus changes in myenteric nerve plexus of stomach tissues, which the neural factor of extreme hypofunction of the stomach..(8) ICC less amount, vacuolus changes and damaged cellular organs are the important pathological characters of DGP rats. (9) Smooth muscle smooth muscle happy atrophy, break, absence, apoptosis, arrangement disorder, muscle fibrosis, vacuolus changes and disruption of myocyte, swelling of mitochondrion, which are the muscle factors of extreme hypofunction of the stomach.. (10) Aglumin can increase blood apparent viscosity, aggravate pancreatic islet damages, induce metabolic disturbance of sugar, grease and free radicals, provocate and aggravate aggravate vascular lesion, reduce the bloodstream of stomach, result in secretion disorder of GHs and neurotransmitter.(ll) Lumbrukinase may lower blood viscosity, improve hypoxia of pancreatic islet and stomach tissues, lessen the damage of pancreatic islet, inhibit the blood vessel of stomach, depress parasecretion of GHs and neurotransmitter, inhibit the ischemia and hypoxia.(12) traditional Chinese drug can induce improve STZ, damage pancreatic islet, lower the concentration of the sugar, grease and free radicals, improve blood rheology, protect blood vessel of stomach form damaging, improve the synthesis and parasecretion of GHs and neurotransmitter, protect neurocytes of stomach, keep ICC and smooth muscle from injuring, improve gastric motility.
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