| ObjectiveAs stomachal blood flow was a key point which ensured gastric motility successfully, we planed to study the effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) which was representative prescription of invigorating the spleen and promoting blood flow methods on enteric nervous system(ENS), interstitial cells of cajal(ICC) and smooth muscle cells(SMC) of sinus ventriculi in diabetic gastroparesis(DGP) rats, so that we might reveal internal mechanism on morbility of DGP and the role of blood flow in the pathogenesy of DGP, and identify the site of action, link and molecule mechanism which "TWK" treated DGP. We wished to develop new methods and theory evidence in the traditional Chinese medicine prevention and cure of DGP, to provid an effective Chinese drugs preparation in clinical therapy of DGP. MethodsOne hundred thirty-one male SD rats who were SPF class were randomly divided into the control group(10 rats), model group(22 rats), ethamsylate group(33 rats), lumbrukinase group(33 rats) and TWK group(33 rats). All rats on an empty stomach, having been fasted for 12 hours, were measured blood sugar and weight in the morning. Except for rats in the control group, surplus rats were injected streptozotocin(STZ) solution(0.1mmol/L) which was prepared freshly by citric acid and citrate sodium buffer solution into their abdominal cavities with a 60mg/kg dosage one time. Rats in the control group were injected citric acid and citrate sodium buffer solution into their abdominal cavities with isasteric dosage one time.After seven days making diabetic mould, we collected every rat' s tail veinblood by shearing tail and measured blood sugar, we looked blood sugar more than or equal to 16.7mmol/L as experimental internalized standard. Rats consistented with experimental internalized criteria began to be treated after 10 days making diabetic mould. Rats in the TWK group were treated by traditional Chinese medicine "TWK" apozem which were given by intragastric adminisreation one time per day. Rats in the ethamsylate group were treated by ethamsylate injection which were given by intragastric adminisreation one time per day. Rats in the lumbrukinase group were treated by lumbrukinase enteric-coated capsules which were injected into their abdominal cavities one time per day. Rats in the control group and rats in the model group were treated by tales doses distilled water which were given by intragastric adminisreation one time per day. Administration dosage was as 2 times as adule clinical common used dose. Animal general states were observed and recorded every day. Each rat was weighed per 3 weeks. Under feeding and drinking free, all rats were feeded by composite granulometric animal feeds and observed for 30 weeks.All rats' blood-fasting sugar were measured on 17 week. After having been observed for 30 weeks, all rats on an empty stomach, having been fasted for 24 hours, were measured blood sugar in the morning, then were given 5ml phenolsulfonphthalein solution(lmg/ml) by intragastric adminisreation. After 30 minutes, we took 6ml blood from each rat' s heart, separated blood serum, and measured contents of FINS. C-P. TC. TG. HDL. LDL. SOD and MDA. We also got 4ml blood from each rat' s heart and measured hemorheology indexes in rat' s plasm. Then we put rats to death by stretching their collum, opened the rats' abdominal cavity, collected gastric residue, obtained optical density value with 722 grating spectropholometer and calculated rats' gastric fluid emptying rate. Subsequently, we got out the stomach and pancreas. Two samples of sinus ventriculi were made HE staining> specific staining (Masson staining> Mallory staining, toluidine blue staining) and immunohistichemistry staining after fixed by 10% neutral formalin solution and 4% paraform solution, dehydrated, embeded by paraffin and sliced, so that we could observe pathological lesion of gastric micrangium, gastric myenteric nerve plexus, gastric neurotransmitter such as SP. VIP. 5-HT. nNOS, gastric interstitial cells of cajal(ICC) , gastric smooth muscle cells(SMC) through light microscope. Other sample of sinus ventriculi was made up electron microscope viewing specimen so as to know pathological lesion of ultrastructure of gastricICC-, SMC> myenteric nerve plexus> micrangium and relation among ICC? SMC and enteric nervous system(ENS) through transmission electron microscope. Samples of pancreas were fixed by 10% neutral formalin solution, dehydrated, embeded by paraffin , sliced , and made HE staining and special staining (aldehyde-fuchsin and orange G) in order that we could know how pancreatic tissue and pancreatic islet were destructed through light microscope. Results1. Establishing rat' s diabetic model and rat' s diabetic gastroparesis model and effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) to themMaking diabetic model ago, all rats' blood sugar level and body weight were not different. After 7 days making diabetic model, mean of rats' blood sugar in the control group was 3. 20mmol/L, and 111 rats' blood sugar in diabetic model group were from 16.72 to 24. lmmol/L, mean was 19.0±l. 65mmol/L Consistented with experimental internalized criteria(blood sugar more than or equal to 16. 7mmol/L) rate was 91. 74%. After 30 weeks, mean of rats' blood sugar and body weight in the control group were respectively 2. 90mmol/L and 523g , FINS , C-P > TC > TG ^ HDL ^ LDL , SOD and MDA were respectively 1176. 75±204. 71pmol/L , 0. 035+0. Olpmol/L ^ 1. 32±0.08mmol/L > 0. 60+0.18 mmol/U 0. 51 ±0. llmmol/U 0. 29+0. 05mmol/U 392. 22±11.48U/ml and 8. 06±0. 54 mmol/ml. Mean of rats' blood sugar and body weight in the model group were 25. 75mmol/L and 192g respectively, FINS^ OP, TC> TG, HDU LDU SOD and MDA were 392. 08 + 125. 91pmol/U 0. 070+0. 024pmol/U 5. 88+0. 33mmol/U 4.37 + 0.30 mmol/U 0.10±0. 05mmol/U 1. 51 + 0.12mmol/U 115.98 + 18. 50U/ml and 17. 23±1. 03 mmol/ml respectively. There were a very significant difference in these biochemical indicators between rats in the control group and rats in the model group(p<0. 05 or p<0. 01). But there were no or few beta cells in rats' pancreatic islets in the model group by HE staining and aldehyde-fuchsin and orange G staining. The result demonstrated that rat' s diabetic model was established successfully.The experimental results indicated that nNOS in rats' gastric myenteric nerve plexus of the model group were obviously not so many as one in rats' gastric myenteric nerve plexus of the control group(p<0.05). Phenol red excretion test result discovered also that gastric fluid emptying rate in rats of the model group were significantly lower than one in rats of the controlgroup(p<0.01). Thus, rats in the experiment had been successfully established into diabetic gastroparesis model.The results showed that after 30 weeks, blood sugar-, body weights FINS> (H\ T(\ TG> HDU LDU SOD and MDA in rats of the lumbrukinase group and ones in rats of the ethamsylate group were significantly difference compared with ones in rats of the control group(p<0.05 or p<0.01), but were no obviously different compared with ones in rats of the model group. There were no or few beta cells in rats' pancreatic islets in the lumbrukinase group and in the ethamsylate group by HE staining and aldehyde-fuchsin and orange G staining. However, in rats of the TWK group the biochemical indicators had amendment, hyperemia of pancreas were lessened through light microscope, and there were more beta cells in rats' pancreatic islets in the TWK group than ones in the model group. The results manifested that "TWK" could improve destruction of beta cells in rats' pancreatic islets which were caused by STZ, and cut down blood glucose also.After 30 weeks, in rats of the TWK group gastric fluid emptying rate was obviously higher, and nNOS in gastric myenteric nerve plexus was significantly more than ones in rats of the model groupN ones in rats of the lumbrukinase group or ones in rats of the ethamsylate group, but were no obviously difference compared with ones in rats of the control group. The results manifested that "TWK" could speed up diabetic rats' gastric fluid emptying. 2. Changes of hemorheology indexes and gastric micrangium pathology in rats of diabetic gastroparesis model and effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) to themHemorheology indexes in rats of the model group were abnormal, such as whole blood viscocity> blood plasma viscocity> hematocrit* reduction viscocity and erythrocyte aggregation fingerzahl were obviously higher , erythrocyte sedimentation rate were quicker, red blood cell deformation fingerzahl were more augmented and f ibrinogen were more augmentative than ones in rats of the control group. Under light microscope, gastric micrangium in rats of the model group were dilated, crudeness or fineness of caliber were different, blood vessel presented distension liking sack, some lumens had deformed and narrowed, basal lamina were obviously thickening, there were a great deal of inflammatory cells infiltrate in mesenchymal, but microvessel count were obviously less than ones in rats of the control group. Through transmission electronmicroscope, we observed that gastric micrangium were 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 rats of the TWK group and in rats of the lumbrukinase group hemorheology indexes took a turn for the better compared with ones in rats of the model group. 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 rats of the ethamsylate group hemorheology indexes were worse, pathological lesion of gastric micrangium were significantly aggratated, and microvessel count were less than ones in rats of the model group through light microscope and transmission electron microscope.3. Changes of gastro enteric nervous system(ENS) pathology in rats of diabetic gastroparesis model and effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) to themIn rats of the model group, number of neuron cell in gastric myenteric nerve plexus were obviously less than ones in rats of the control group (p<0. 01), amount of tigroid body grana in neuron were also lessened and presented obviously lysis or loss through light microscope. Under transmission electron microscope, in rats of the model group number of neuron cell in gastric myenteric nerve plexus obviously lessened and number 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. In rats of the model group, SP, 5-HT and nNOS in neurotransmitter were obviously less in gastric myenteric nerve plexus, but VIP were significantly more than ones in rats of the control group (p<0.05^p<0.01).Through number of neuron cell in gastric myenteric nerve plexus also lessened in rats of the TWK group and in rats of the lumbrukinase group, these tooka turn for the better compared with ones in rats of the model group* ones in rats of the TWK group were better (p<0.01), but ones in rats of the ethamsylate group were least. Loss and lysis of tigroid body in neuron after treatment of "TWK" or lumbrukinase were obviously lessened. Damage of tigroid body in neuron after treatment of ethamsylate became more serious.After treatment of "TWK", SP^ 5-HT and nNOS in neurotransmitter increased, and VIP reduced in gastric myenteric nerve plexus. SP> 5-}fi\ VIP and nNOS in neurotransmitter were not marked change in gastric myenteric nerve plexus after treatment of lumbrukinase or ethamsylate. We also found that ultramicrostructure of rat' s gastric myenteric nerve plexus were obviously improved after treatment of "TWK" or lumbrukinase, but pathological lesion of ultrastructure of rat' s gastric myenteric nerve plexus were aggratated after treatment of ethamsylate.4. Changes of gastro smooth muscle pathology in rats of diabetic gastroparesis model and effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) to themUnder light microscope, we found that in rats of the model group endochylema of gastro smooth muscle cells were tasteless dyeing, and were lucency, there were apomorphosis liking vacuole, gastric ring muscle and longitudinal muscle became thin, longitudinal muscle were especially obvious, but observed that gastro smooth muscle were fibrous degeneration. We also found that the arrangement of smooth muscle cells were disordered, cytoplasm in muscle cells were abundantly dissolved, mitochondria were tumescent% liking vacuole chang and even lysis in rats of the model group through transmission electron microscope. We discovered that apoptotic index of SMC in rats of the model group were 24. 33±2. 34% with TUNEL method, there were significantly difference compared with rats of the control group(p<0.0l). Protein expression of Bcl-2 in SMC were reduced by immunohistichemistry staining in rats of the model group, but protein expression of Fas were overexpression, there were significantly difference compared with rats of the control group(p<0.01).However, pathological lesion of gastro smooth muscle were obviously improved in rats of the TWK group and in rats of the lumbrukinase group. They reflected principally that the arrangement of smooth muscle cells were in good order, there were few vacuoles, fibrous degeneration of gastro smooth muscle were amelioration, and apoptotic index of SMC were obviously higher than ones inrats of the control group(p<0.01), but they were significantly lower than ones in rats of the model group(p<0.01), cells of protein expression of Fas in SMC were significantly fewer than ones in rats of the model group(p<0.01), but cells of protein expression of Bcl-2 were oviously more than ones in rats of the control group(p<0.01) under light microscope. Compared with rats of the model group, the arrangement of smooth muscle cells were regularity, there were few big dissolubility vacuoles in cytoplasm of muscle cells, engorgement of mitochondria were not obvious through transmission electron microscope in rats of the TWK group and in rats of the lumbrukinase group. But in rats of the ethamsylate group pathological lesion of gastro smooth muscle made more serious. They showed that endochylema of gastro smooth muscle cells were tasteless dyeing, and were lucency, there were apomorphosis liking vacuole and even collapse, gastric ring muscle and longitudinal muscle became thinner, fibrous degeneration of gastro smooth muscle were more severe, apoptotic index of SMC grew in number, cells of protein expression of Fas in SMC increased, cells of protein expression of Bcl-2 reduced through light microscope. Under transmission electron microscope, the arrangement of smooth muscle cell were chaotic, there were many big dissolubility vacuoles in cytoplasm of muscle cells, mitochondria were obviously tumescent, liking vacuole chang or lysis.5. Changes of gastro interstitial cells of cajal(ICC) pathology in rats of diabetic gastroparesis model and effect of interfering in blood flow medicine and "Tang-Wei-kang" (TWK) to themOur research discovered that number of gastro interstitial cells of cajal (ICC) in rats of the model group were significantly fewer than ones in rats of the control group by applying c-kit antibody immunohistichemistry staining detection method under light microscope. So' through transmission electron microscope we found that in rats of the model group the construction of gastro interstitial cells of cajal connection were damaged, number of cell connection between ICC decreased, rough endoplasmic reticulum in ICC expanded, there were vacuolization in intracytoplasm of ICC, number of mitochondria in ICC lessened, mitochondria swelled% dissolved or vacuolated. In rats of the TWK group and in rats of the lumbrukinase group , pathological lesion of ICC was palliated. They displayed that number of ICC increased obviously, pathological lesion of connection between ICC> ICC and SMC> ICC and nerve terminal werealso improved, their construction was clear and compact. Nevertheless, pathological lesion of ICC was most severe in rats of the ethamsylate group. They showed that number of ICC were least, gap junction between ICC ICC and SIC ICC and nerve terminal diminished significantly, construction of remaining cell-cell junction was also demolished, their structure was not clear, their conjunction was loose. Conclusionl.The experimental result presented that there was determinatus relation between transmutation factor of blood stream and pathogenesy of diabetic gastroparesis, but it is not major nosogenesis. Improving microcirculation might achieve to promote gastro dynamic effect in therapy.2. We presumed that pathogenesy of diabetic gastroparesis was possiblely as follow: When diabetes took place, glycometabolish disorder % hyperlipemiaand plasma fibrinogen increasing were caused, so abnormal hemorheology was induced, it evoked capillary vessel wall of gastrointestinal tract thickened, structure demolished, stasis of blood stream and gastrointestinal tract ischemia, ischemia of interstitial cells of cajaK cellula nervosa> smooth muscle cells and mucous membrane cells were caused, and energy supplyed reduction or energy lacked, damage of cellular membrane system-. plasmolysis% engorgement of mitochondria and apomorphosis liking vacuole were resulted in, thus function of the cells were acutely damaged and demolished balance of many kinds of neurotransmitter in myenteric nerve plexus, and urged occurrence of gastrointestinal complication in diabetes. Pathological change and disfunction of basic function unit of gastric motility were possibly critical local mechanism while diabetic gastric dyskinesis was took place.3. The finding illustrated that "Tang-Wei-kang" (TWK) could cut down blood sugar and promote gastric dyskinesis, and was a Chinese drugs pharmaceutics which had certainly therapeutic effect. We supposed that treating mechanism of "TWK" in diabetic gastroparesis was possiblely as follow: through lossing blood sugars lowering blood fat> improving hemorheology> adding volume of blood flow, pathological changes of capillary vessel> ICC> cellula nervosa and SMC in gastrointestinal tract were improved , effect of local excitatoty neurotransmitter such as SP and 5-HT in gastrointestinal tract were raised, but effect of local inhibitive neurotransmitter such as VIP or NO in gastrointestinal tract were degraded, pathological change and disfunction ofbasic function unit of gastric motility were recovered, so that "TWK" could achieve to advance the effect of gastrointestinal motility.
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