| ObjectiveThe paper studied and researched the effect of blood flow interfered in medicine with blood coagulation Ethamsylate , promoting blood flow , Lumbrukinase and the traditional Chinese medicine "Tang wei kang" to diabetic rats gastric nerve, gut hormone and related gastric mucosa immunological regulation each other, then identified the role of blood flow factor and neuroendocrine-immune network in diabetic gastric motility disorder pathogenesis, illuminated the effect of reinforcing spleen and promoting blood flow representative prescription "Tang wei kang" to diabetic gastric motility disorder rats neuroendocrine-immune network. To reveal diabetic gastric motility disorder pathogenetic internal mechanism and and make its clear site, link and molecule mechanism of action of "Tang wei kang" treated diabetic gastric motility disorder, to establish new method and based on theory and to provide an effective Chinese drugs pharmaceutics for clinical therapy. Material and methodsSD SPF class rats were weighted 160±20g and randomly divided two groups : normal groups 10 rats and mould groups 121 rats, all rats were fasted 12h then tested blood glucose mane sequenti in empty stomach, except high blood glucose or deviated normal number and upper number rats, weighted, peritoneal injected made up streptozotocin(STZ)(0.1mol/L)freshly by sodium citric buffer once time, injected dose 60mg/kg , normal control groups rats were peritoneal injected the same dose 0.1mol/L(pH4.5) sodium citric buffer only, the seventh mould day all rats were tested blood glucose, blood glucose was higher 16.7 mmol/L as success for making mould.According to the computer successed mould rats were divided mould group(30), ethamsylate group(20), lumbrukinase group(30) and the traditional Chinese medicine group(30). From the beginning of the thrid successed mould, the traditional Chinese medicine group rats were lavaged "Tang wei kang" decoction 1g/ml;ethamsylate groupwas given ethamsylate with intragastric administration and lumbrukinase group with peritoneal injection;mould group and control group were lavaged with the same dose distilled water, the three groups were given medicine per week six time, medicine was not given on Sunday. Given drug dose was twice than adult clinical dose.To observe and record common condition of rats including drink and foods egestions excretions hairsN mental states automatic actions cataracts death etc.) and weigh body weight one time per week, according to the new weight to caculate drug dose. The seventeenth and the thirtieth week were tested blood glucose in empty stomach, then gastric emptying was tested to sure diabetic gastric motility disorder rats successed mould.The thirtieth week, collected blood from rats and measured insulins C-peptide with radio-immunity, determined four items of blood fat with blood biochemistry analysis apparatuss SOD and MDA, blood plasma was tested hemorheology, then put rats to death and selected its end part of pancreatic island to fix with neutral formalin, to embed and slice then stain hematoxylin-eosin(HE dyeing) and special aldehyde-fuchsin-orange G for observation of impaired pancreatic island;tested Helicobacter pylor with reagent paper before getted sinus ventriculi to fix with neutral formalin, to embed and slice then was observed with HE dyeing to show gastric mucosals muscle layers microvascular and leukomonocyte pathological changes, furthermore with toluidine blue for mast cellss with methyl green-pyronin for plasma cell and with Masson method for nerve fibre, then with immunohistochemistry dyeing to detect gastric mucosa apoptosis gene Bcl-2s Fass inflammatory factor tumor necrosis factor-a (TNF — a) > interleukin-lbeta (IL-1 P ) s neurotransmitter 5-hydroxytryptamine (5-HT) snitricoxide synthase 1 (NNOS) sC-kit factor in ICCsthe vascular endothelial cell superfic receptor, TUNEL in situ hybridization showed gastric mucosa apoptosis gene;sinus ventriculi was fixed with 4% paraformaldehydes embedded and sliced to detected gut hormone substance P (SP) and vasoactive peptide (VIP) with immunohistochemistry, the last body of stomach fixed with glutaraldehyde, then ultramicrostructure was observed gastric microvascular and nerve fibre with transmission electron microscope (TEM), the same time chosed thymus gland and spleen to weigh. Results1 The results of making diabetic gastric motility disorder rat' s mould and affect of interventional medicine 1.1 The changes of body weight and pancreatic glandAll group weight was no significant difference before mould (P>0. 05) , the third, the sixth, the ninth, the twelveth, the fifteenth, the eighteenth, the twenty-one, the twenty-fourth, the twenty-seventh and the thirtieth all groups(mould group, ethamsylate group, Lumbrukinase group and Traditional Chinese medicine group) were weighed low to normal group obviously (P<0. 01), all groups(mould group, ethamsylate group, Lumbrukinase group and Traditional Chinese medicine group) weighed without significant difference each other(P>0. 05). All model groups pancreatic gland and islet cell were impaired with different degree, islet cell was atrophy and disorder, pancreatic gland swelled, the count of islet 6 and a cell was decreased markedly, each medicine group was compared with significant difference each other (P<0. 05) .1.2 The changes of blood sugar and serumal insulin and C-peptide Before diabetic model, all rats' blood sugar level and body weight were not different. After 7 days making diabetic model, the mean blood sugar of the control group rats was 3.20mmol/L, and 111 diabetic model group rats were from 16.72 to 24.1mmol/L, the mean blood sugar was 19.0±1.65mmol/L. Consistented with experimental internalized criteria (blood sugar more than or equal to 16.7mmol/L) rate was 91.74%. After 30 weeks, the mean blood sugar and bodyweight of the control group rats were respectively 2.90mmol/L and 523g, DN^ C-P were respectively 1176.75±204.71pmol/I^ 0.035±0.01pmol/L. The mean of the model group rats were 25.75mmol/L and 192g respectively, INSn C-P were 392.08±125.91pmol/LN 0.070±0.024pmol/L> respectively. There were a very significant difference in these biochemical indicators between the control group rat and the model group rat (P<0.05 or P<0.01).The changes of above-mentioned items were synthetized and showed the diabetic mould rat establishment. Tested gastric emptying showed all mould rats was lower than the control group with marked difference (P<0. 01), illustrated gastric motility disorder mould made and medical group without significant difference each other (P>0.05) . 2 The result of hemorheoIogy and gastric mucosal apoptosisAfter the diabetic mould was the thirtieth week, all mould group rats(mould group ^ Ethamsylate group > Lumbrukinase group and traditional Chinese medicine group) hemorheology indexes in the model group rats were abnormal, such as whole blood viscocity> blood plasma viscocity> hematocrit% reduction viscocity and erythrocyte aggregation fingerzahl were obviously higher, erythrocyte sedimentation rate were quickejv-— red blood cell deformation fingerzahl were more augmented and fibrinogen wrac more augmentative than ones in the control group rats. /After 30 weeks, mean of the control group rats' TCU TG> HDL> LDL^ SODabd MDAwere respectively 1.32±0.08mmol/L , 0.60±0.18mmol/L ^ 0.51±0.11mmol/L , 0.29±0.05mmolO^ 392.22±11.48U/ml and 8.06±0.54mmol/ml. Mean of the model group rats' TC> TG , HDL ^ LDL > SOD and MDA were 392.08±125.91pmol/L* 0.070±0.024pmol/L ^ 5.88±0.33mmol/L > 4.37±0.30mmol/L ^ 0.10±0.05mmol/L ^ 1.51±0.12mmol/L> 115.98±18.50U/ml and 17.23+1.03mmol/ml respectively. The mean of Ethamsylate group> Lumbrukinase group and the traditional Chinese medicine group TC> TG> HDL, LDL, SOD and MDA were 5.38+0.36mmol/U 4.14+0.21 mmol/U 0.22 ±0.09mmol/U 1.56±0.07mmol/U 93.86±18.18U/mK 17.45±0.64mmol/ml;5.22 + 0.21mmol/U 3.76+0.41mmol/U 0.25+0.13mmol/U 1.52±0.11mmol/U 126.50+10.00 U/mK 12.46±0.72mmol/L: 4.39+0.48mmol/U 3.66±0.29mmol/L, 0.31+0.07ranol/L> 1.26+0.22mmol/U 247.46+21.47U/mK 10.24±0.43 mmol/ml respectively. All mould groups were compared with the control group and, compared with mould group and Ethamsylate group, showed Lumbrukinase group and the traditional Chinese medicine group showed marked difference(P<0.01).2.1 The pathological change and results of cell apoptosis expressed about gastric mucosaGastric mucosa was tested by Helicobacter pylori paper and showed negative, mould group gastric mucosal cell disordered, mould group was serious and bleed under light microscope with H.E staining. Gastric mucosa cell apoptosis was tested and showed Ethamsylate group cell apoptosis was higher than the other mould groups, the second was mould group, then Bcl-2 expressed the highest in control group, the other mould groups was opposite to above-mentioned result.3 The changes of diabetic gastric moti I ity disorder neuro-endocrine-immunity 3.1 The expressed results of gut hormone SP> VIP> neurotransmitter 5-HTn nN0S> and the pathological changes of nerveThe positive SP and VIP expressed in gastric mucosa and muscle plexus, its regulated gastric motility contrarily, the positive SP expressed significant difference with the other experimental group (P<0.01) , then the positive VIP was significant high (P<0.01) . Lumbrukinase and the traditional Chinese medicine group had not difference(P>0.05), the other groups had significant difference each other (P<0.01) .In the model group rats, number of neuron cell in gastric myenteric nerve plexus were obviously less than ones in the control group rats(P<0.01), amount of tigroid body grana in neuron were also lessened and presented obviously lysis or loss, image analysis displayed that gray scale value was higher through the optical microscope. Under transmission electron microscope, thecell population of neuron cell about the model group rats in gastricmyenteric nerve plexus obviously lessened and the cell population of non-neuron cell increased, neuronic nucelus in myenteric plexus appeared lysis and necrosis, some kytoplasm were dissolved, mitochondria were tumescent, cristae resolved, rough endoplasmic reticulum expanded, some entosarc dissolved, some neural axon and dendron acra were tumescent or deliquescent, electron density were unevenness, some dilated into vacuole obviously, synaptic vesicle content in neural varicosity obviously lessened. Neurotransmitter 5-HT expressed in the gastric bottom and central region and expressed nNOS in the muscle plexus was significant high compared with the other experimental group (P<0.01) , the other mould group was significant difference each other (P<0.0l) . 3.2 The changes of TNF-a ? IL-1 {3 > immune organ^ immune eel IThe weight of all mould groups immune organ thymus gland and spleen was low, lymph cell and mast cell of control group was significant lower than the other mould groups, mould group infiltrated lymph cell, mast cell released grains from mould group;TNF-a expressed in gastric mucosa of control group and was lower than the other experimental group ( P<0.01), the expressed TNF-a of Ethamsylate group was higher than model group markedlyC P<0.01), and the positive express decreased in the traditional Chinese medicine "Tang wei kang" and Lumbrukinase group, but IL-1B expressed negative. Tne medulla of thymus gland about all model groups was major injury, the structure of thymus corpuscles was unsharpness and thymus gland was apomorphosis and analosis.the thymus corpuscles of the traditional Chinese medicine "Tang wei kang" and Lumbrukinase group was clear but the part of its had apomorphosis;the splenic corpuscle numbers of the spleen about model group and Ethamsylate group decreased and the volume of splenic corpuscle contracted, the structure of splenic corpuscle was chaotic and the limit was unsharpness;the splenic corpuscle of the traditional Chinese medicine "Tang wei kang" and Lumbrukinase group had not unsharp structure by optical microscope with H.E staining.3.3 The changes of microvascular endotheiial cell and ultramicrostructure about microvascularThe cell population of microvascular endotheiial cell for the control group was 3.30±0.90, the model group > Ethamsylate group % Lumbrukinase group and the traditional Chinese medicine "Tang wei kang" group was significant high than the control group (P<0.01);the cell population of Ethamsylate group was higher than the model group markedly (P<0.01), the cell population of Lumbrukinase group and the traditional Chinese medicine 'Tang wei kang" group was significant high than the model group and lower than the Ethamsylate group markedly (P<0.01), then the traditional Chinese medicine "Tang wei kang" group was lower than the Lumbrukinase group significantly (P<0.01).The percentage of areas and the average optical about CD34 in Anicrovas^ularendothelium for the model groupn Ethamsylate group^ Lumbrukinase group and the traditional Chinese medicine "Tang wei kang"was lowest than the control group markedly ( P<0.05 ) , the average gray scale was highest than the control group significantly, then the three items of the traditional Chinese medicine "Tang wei kang" was higher than the model groupn Ethamsylate group-. Lumbrukinase group markedly (P<0.01) .Through transmission electron microscope, we observed that gastric capillary vessel was deformed and narrowed, endotheliocyte were edema, intracytoplasm mitochondria were tumescent and degenerative, cristae were short and lost or vacuolization, nuclear chromatin assembled and kept to the side, electron density in basal lamina were unevenness, construction were dim and thickening, micrangium lumens waxed, vessel wall were complete and no disrupt. In the traditional Chinese medicine "Tang wei kang" group rats and the Lumbrukinase group rats hemorheology indexes took a turn for the better compared with ones in the model group rats. So under light microscope and transmission electron microscope, we found also that pathological lesion of gastric micrangium were obviously lessened, microvessel count were significantly incremental. Nevertheless, in the ethamsylate group rats hemorheology indexes were worse, pathological lesion of gastric micrangium were significantly aggratated, and microvessel count were less than ones in the model group rats through light microscope and transmission electron microscope. Cone I us i on1.1 The gastric emptying was tested for diabetic rats and showed the diabetic rats were gastric motility disorder.1.2 The traditional invigorate the spleen and promoting blood flow Chinese medicine "Tang wei kang" protected gastric mucosa of diabetic rats.1.3 The immunity of diabetic gastric motility disorder rats and correlation factor nerves and gut hormone were abnormality;they correlated with gastric motility functional unit directly and indirectly. Immunity and neurohormone composed neruohormone-immune net to regulate gastric motion each other. |
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