| Objective: The slow transit constipation is thought as a primary defect that the movement of contents from the proximal to the distal colon and rectum is slower than that of normal. However, the pathophysiology of slow transit constiption is still not delineated. Rhubarb is the traditional Chinese medicine, it has noticeable effect of causing decanta and promoting intestinal enterocinetic, but it's concrete mechanism is unclear. In order to approach the mechanism of rhubarb. we establish the model of slow transit constipation by feeding rats with Diphenoxylate, study the pathogenesis of slow transit constipation by investigating the changes of interstitial cells of Cajal, enteric nerves system and colonic myoelectric activity in rats; then study the occurrence mechanism in the protein level, deliver the methods of prevention and cure for STC and study the new medicine for STC by sieving the function target.Methods: Establish slow transit constipation model in rats by Diphenoxylate. Then the rats were randomly divided into two groups, one group given the dry foodstuff without any drug and the other group given one containing Rhubarb , 480mg/kg.After 40 days ,colonic myoelectric activity was recorded and the time of colonic transit was measured by feeding rats the active carbon .At last the rats were killed, the colonic tissues (1cm apart from cecum and 2cm apart from anus) were dyed with Hematoxylin and eosin (HE). Cholinergic nerves and nitrergic nerves were investigated in colonic myenteric plexuses in the rats with histochemistry and immuneohistochemistry, and interstitial cells of cajal were investigated in colonic myenteric plexuses with ZIO histochemistry in the rats. Weight 0.3g the colonic tissues(wiped off chorion) and whet them under the condition of liquid nitrogen. Then distill the proteins of the colonic tissues by adding proteinum lysate with 1ml to 100mg proportion and separate them in two dimensional gel electrophoresis. After dyeing the proteins with Coomassie light blue and discoloring them ,analyze the proteins with Mass spectrum to get the data of their sequence and search them in nr's databank with software of Mascot.Results: (1) The movement of contents from the proximal to the distal colon and rectum in model rats was significantly slower than that of normal rats. In model rats group, the time of the first black faeces was 357±24min, it was longer than that of normal rats (241 ±37 min, p < 0.01).By contrast, in the group given Rhubarb, it was significantly faster than that of STC rats (255 ± 15min, P<0.01 = .(2) The frequency of colonic slow wave In STC model rats showed two-way alteration. It was very clear that the frequency of colonic slow wave increased in the group treated by Rhubarb (8.62±1.20/min vs 5.60 + 1.20/min, p<0.01) and amplitude decreased (0.33±0.05mv vs 0.46 + 0.05 mv, p< 0.01) .At the same time, when STC model group with faster frequency were given Rhubarb ,the frequency of colonic slow wave significantly decreased(23.21 ±3.86 /min vs 30.23 ±3.86/min, p < 0.01) and amplitude varied (0.19±0.03 mv vs 0.20 + 0.03 mv, p< 0.01). (3) The chronic inflammation occurred in colonic mucosa of all model rats. In the colonic myenteric plexuses of rats with slow transit constipation, cholinergic nerves and cholinergic cells were significantly decreased with denser immunostainning, but its' volume increased. After treated by Rhubarb ,cholinergic nerves and cholinergic cells tended to be normal(positive cells were 25.71±5.36 vs STC model group 14.32±4.60 p < 0.01 -, the ratio of nuclei/cytoplasm was 0.71±0.218vs STC model group0.58±0.19, p<0.01). In model group, nitriergic nerves was significantly reduced, nitriergic cells decreased and had lighter immunostainning .After treated by Rhubarb ,nitriergic nerves was significantly increased (positive cells werell.76±2.31 vs STC model group 7.10±2.04, p<0.0l; lightly positive cells were 0.62±0.45 vs STC model group 2.21 ±1.38, p < 0.01). In the colon of rats with slow transit constipation, interstitial cells of Cajal(ICCs) were not well-distributed, the number of ICCs in some area increased but decrased in other area. Nervous processes could not connect well to be a network. But In the group treated by Rhubarb, ICCs was well-distributed as similarly as to the normal rats and nervous processes connected so well as to form a network. (4)Variances of protein expression of rat colon under different conditions were analyzed by two-dimensional gel electrophoresis. The number of protein spot was 256, 293,247 respectively in normal rats, STC rats and rhubarb-treated rats. 53 different spots were found between normal and STC rats, 37 between rhubarb-treated and STC rats,21 between normal and rhubarb-treated rats.(5)different spots were identified by MALDI-TOF-MS. Obvious distinction wasobserved in the genesis and treatment of STC, such as follows: mast cell protease( A1), non-specific dipeptidase(A2), mitochondrial aldehyde dehydrogenase precursor(A3), Calponin(CaP, Bl), histone H2B(B2), similar to protein RAKb (B3)o The expression of AK A2, A3 up-regulated in the gel graph of rhubarb-treated rats, however, the expression of BK B2> B3 down-regulated in the gel graph of rhubarb-treated rats.(6) Calponin is correlated with the gastrointestinal movement, the amount of Calponin lowers when the movement strengthens, vice verse. Mitochondrial aldehyde dehydrogenase which is closely correlated with the energy metabolism of cells and mast cell protease which is closely correlated with the immunological reaction of colon mucosa are down-regulation in rat colon.Conclusions: 1 .Pathological changes of colonic myenteric plexuses occured in rats with slow transit constipation,dysfunction of colonic transit is close related with the changes of colonic myenteric plexuses. Abnormality of distribution and numbers of interstitial cells of cajal is responsible for colonic motility abnormality. 2. Abnormality of colonic myoelectric activity may be a reason of slow transit constipation. Rhubarb can adjust the abnormal frequency and wave of slow wave in STC rats. 3. Obvious variances of protein expression were found by analyzing the graph of the two-dimensional gel electrophoresis among normal, STC and rhubarb-treating rats. 4. The down-regulation of mitochondrial aldehyde dehydrogenase > mast cell protease as well as non-specific dipeptidase in rat colon suggests the functional impairment of the oxidoreduction of mitochondrion are very important in the genesis and development of STC. The immunological reaction of STC rats weakens, the function of digesting and absorbing protein may be damaged to some extent.5. Calponin plays a vital role in regulating the gastrointestinal movement and maybe mediate the effect of gastrointestinal dynamia of rhubarb . It may regulate the gastrointestinal movement by changing its amount. |
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