| Objective To observe the morphological changes in the network of enteric nerve-interstitia cells of Cajal (ICC)-smooth muscle in rats with Yang Ming Fu Shi Syndrome, and to investigate the therapeutic effect of purgative therapy on the function of gastrointestinal motility.Methods One handred Wistar rats were randomly divided into three groups:control group (n=20), Yang Ming Fu Shi Sydrom model group (n=40) and Da Cheng Qi decoction treated group (n=40). The rats of control group were injected 1.0ml of normal saline into the peritoneal cavity. The rats of Yang Ming Fu Shi Syndrome model group were perfused chinese herb of the hot and itself dung, then 1.0ml suspension of Escherichia coli was injected into the peritoneal cavity. The rats of Da Cheng Qi decoction treated group(DCQT group) were administrated by gavage with Da Cheng Qi decoction (twice a day,each one 1.0ml/100g) after the Yang Ming Fu Shi Syndrome was established. After five days, the proximal 10cm segment of jejunum was taken and the whole-mount preparation from each group were studied used c-Kit, vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), substance P(SP) and vasoactive intestinal peptide (VIP) immunofluorescence double-staining with confocal laser scanning microscopy. The expression of protein Cx43 in gap junction between ICC and smooth muscle cell with immunohistochemical analysis was observed, and the morphologic changes in the network of enteric nerve-ICC-smooth muscle was studied with transmission election micros-cope. Results In control group, morphological of gastrointestinal tract was normal, and the colour was pink, there was no adhesion each other, motility funtion was normal.. In model group, gastrointestinal tract was significantly distended, there were much gas and contents in gastrointestinal tract, serosa was congestive and edema; there were bleeding point on great omentum and exudate in peritoneal cavity, inflammation in peritoneal cavity was exited. Many intestinal were gray, mesangial hypertrophy; many liquit content like dung exited in gastrointestinal tract, motility funtion of gastrointestinal tract was significantly decreased. In DCQT group, there were less or no content in gastrointestinal tract. Intestinal was pink, no edema, and motility function recoveried.Observion with confocal laser scanning microscopy In control group, ICCs constituted network in circular muscle layer, longitudinal muscle layer and the myenteric plexus. Enteric nerve of VAChT, nNOS, SP and VIP constructed network each other, and there were many ganglions. ICCs distributed arround the nerve of VAChT, nNOS, SP and VIP, and formed network. The long synapse of ICC distributed with smooth muscle parellely, and short synapse connected each other and formed tight junction with nerve and muscle.In the model group, the number of ICC were significantly decreased compare with control group(p<0.01), the network was disrupted, fluorescence intensity of ICC decreased. The number of nerve fiber of VAChT, nNOS, SP and VIP were significantly decreased compare with control group(p<0.01), nerves network disordered, each connection decrea-sed, fluorescence intensity of enteric nerve decreased(p<0.01). The conne-ction of ICCs and enteric nerves were loosen or disappeared. ICC-enteric nerve network was significantly disrupted.In DCQT group, the number of ICC were significantly increased (p<0.01) compare with control group, the network was recovered, fluore-scence intensity of ICC increased. The number of nerve fiber of VAChT, nNOS, SP and VIP were significantly increased (p<0.01), the network was recovered, fluorescence intensity of nerves increased (p<0.01). The conne-ction of ICCs and enteric nerves were approaching normal, ICC-enteric nerve network was significantly recovered. Expression of protein Cx43 by immunohistochemistry In control group, positive expression of protein Cx43 was distributed in most of cells of longitudinal muscle layer, less of cells distributed circular muscle layer expressed weak postively, and the cells which were distributed in myenteric plexus expressed postively. In model group, postive expression in muscle layer of small intestine was significantly decreased compared with control group (p<0.01), especially in longitudinal muscle layer. In DCQT group, postive expression in muscle layer of small intestine was significantly increased compared with model group (p<0.01), especially in longitudinal muscle layer.Obsercation with transmission electron microscopy The main characteristic of ICCs in control group were abundant mitochondrion, endoplasmic reticulum and mature Golgi body in cytoplasm and synapse; vacuole in falled membrane; many intermediate filaments, no thick filaments; approximate three class tractus; many gap junctions were formed between ICCs and neuron cells, between ICC and myocyte,and among ICCs. The distance of tight junction between nerves and smooth muscle was100nm, and form morphological basic unit:nerves-ICC-smooth muscle.In model group, the synapse of ICC were decreased or disappeared, many nerves swelled; accompanied neurotransmitter vacuole to decreased or disapperaed; granular reticulum of smooth muscle distended, Golgi body swelled, appeared many vacuole because of membrane was in falls; the gap junction between them disappeared; synapse-like junction between ICCs and nerves disappeared, and distance between them was larger; the gap junction between ICCs and smooth muscle disappeared, and distance between them was larger, morphological basic unit of nerves-ICC-smooth muscle was significantly disrupted.In DCQT group, the synapse of ICC was sligherly damaged, nerve ending were abundent, there were less amiunt of swelling nerves, The number of neurotransmitter vacuole was not obviously decreased. The damage in smooth muscle cells was not obvious, synapse-like junction between ICC and nerve was maintenance; the gap junction between ICC and smooth muscle cell was maintained, the distance of gap junction was smaller; There was a little of ligaite substance in tissue intertice, The basic morphology of the unit of nerve-ICC-smooth muscle was maintenanced.Conclusion Gastrointestinal tract was distended in Yang Ming Fu Shi Syndrome rats. Function of gastrointestinal motility was significantly damaged; and the function was recoveried after Da Cheng Qi decoction treatment.In Yang Ming Fu Shi Syndrome rats, number of nerve fiber of VAChT, nNOS, SP and VIP were significantly decreased; and number of ICC were decreased too, enteric nerve-ICC network was disrupted; after treatment with Da Cheng Qi Tang, the numbers of nerves and ICC were increased, and network was recoveried.In rats with Yang Ming Fu Shi Syndrome, the postive expression of protein Cx43 of gap junction in network of enteric nerve-ICC-smooth muscle was decreased, the distance between gap junction was larger. Signal pathway in network of enteric nerve-ICC-smooth muscle was damaged; after treatment with Da Cheng Qi Tang, gap junction and signal pathway was recoveried.
|
PDF Full Text Request