| Colonic dysfunction is a frequent complication of diabetes mellitus(DM). Even though autonomic neuropathy, gastrointestinal hormone andvasodilation are regarded as the important factors in colonic dysfunctionof DM, there are many problems without clarification, while efficaciousremedies need to be found. Recently endogenous carbon monoxide (CO),which is produced by the breakdown of heme catalyzed by hemeoxygenase (HO), has been suggested to play an important role in thegastrointestinal motility as an inhibitory neurotransmitter. Interstitial cellsof Cajal (ICC), which can generate and conduct slow waves, areintermediaries in the transmission and function of the neurotransmitters.It has been recognized recently that HO and ICC play important roles inthe pathogensis of colonic dysfunction with slow transit, whilecorresponding research will be helpful to the invention of new efficaciousremedies.Aim:1. To investigate the role of interference in HO on colonic dysfunction ofdiabetic rats.2. To investigate the role of interference in HO on colonic ICC of diabeticrats.3. To investigate the colonic immunocolocalization of the c-kit andHO-2/HO-1 after the interference in HO.Methods:1. DM model was established by intraperitoneal injection of streptozotocin (STZ) in Sprague-Dawley rats. Six weeks later, thediabetic rats were validated to be suffered with gastrointestionaldysfunction by charcoal propulsion experiment. Then the rest of ratswere randomly divided into 4 groups, named group A(normal control),B(diabetic rats without interference), C(diabetic rats administrated withHemin, the inducer of HO) and D[diabetic rats administrated with zincprotoporphyrinⅨ(ZnPPⅨ), the inhibitor of HO]. The administrationof Hemin and ZnPPⅨpersisted for 3 weeks. The weight and bloodglucose of the rats were tested.2. On the weekend of the ninth week, samples from proximal and distalcolon were obtained. The level of HO were detected by Western blot.The distribution of HO were observed by immunohistochemistry andthe area of HO positive cells were counted. The aim of this step was tovalidate the effect of the interference.3. The GI propulsion rate was tested by the charcoal propulsionexperiment.4. The motilities of the smooth muscle strips isolated from the proximaland distal colon were tested by contraction experiments. Theirspontaneous contraction frequencies, amplitudes and reaction toacetylcholine (Ach) were recorded.5. Samples from proximal and distal colon were obtained. The level ofc-kit expression were detected by Western blot. The distribution of ICCwere observed by immunohistochemistry and the area of c-kit positivecells were counted.6. Using c-kit and HO-2 antibodies or using c-kit and HO-1 antibodies,double immunostaining was carried out. Immunocolocalization wasdetected by means of confocal laser scanning microscopy.Results:1. Six weeks after the establishment of DM model, the gastrointestinal (GI) propulsion rate of diabetic rats were significantly lower than thenormal control (P＜0.05).2. There were no significant difference in body weight or blood glucoseamong the diabetic rats of group B, C and D (P＞0.05). But incomparison with that of the controls, body weight of these 3 groups ofdiabetic rats were significantly delined (P＜0.05), and blood glucose ofthem were significantly raised (P＜0.05).3.①As far as HO-2 expression and the area of HO-2 positive cells,there were no significant difference in those of proximal colon amongthe diabetic rats of group B, C and D (P＞0.05) after the interference.But in comparison with those of the controls (group A), those of these3 groups of diabetic rats were significantly delined (P＜0.05). Therewere no significant difference in those of distal colon among group A,B, C and D (P＞0.05).②As far as HO-1 expression and the area of HO-1 positive cells, thecolonic expression of those were not significant different betweengroup A and B (P＞0.05), but they were markedly higher in group Cthan those in the former two groups (P＜0.05). The expression of HO-1or the HO-1 positive cells could hardly be found in group D.③HO staining was observed by immunohistochemistry in the coloniccells which mainly distributed in the myenteric plexus of proximal anddistal colon. HO immunoreactivity could also be observed insubmucosal plexus and rarely in intramuscular areas.4. After the interference, the GI propulsion rate of group B wassignificantly declined compared to that of the controls (group A)(P＜0.05). In comparison with that in group B, the GI propulsion ratewas dramatically declined in group C (P＜0.05), while that in group Dwas markly improved (P＜0.05), which showed no significant different with that in group A (P＞0.05).5. After the interference, the results of contraction experiments includingspontaneous contraction frequencies, amplitudes and reaction to Achwere significantly declined in group B compared to those of thecontrols (group A) (P＜0.05). In comparison with those in group B,most of the results were dramatically declined in group C (P＜0.05),while those in group D were markly improved (P＜0.05).6. After the interference, the c-kit expression and the area of c-kit positivecells of group B was significantly declined compared to those of thecontrols (group A) (P＜0.05). In comparison with those in group B, thec-kit expression and the area of c-kit positive cells were little declinedin group C (P＞0.05), while those in group D were markly improved(P＜0.05), which showed no significant different with that in group A(P＞0.05). It was observed by immunohistochemistry that thedistribution of c-kit immunoreactivity was in accordance with that ofHO immunoreactivity.7. After the interference, double immuofluorescence displayed that c-kitpositive cells and HO-2/HO-1 positive cells were expressed in identicalsites in the colonConclusions:1. Six weeks after the establishment of DM model, these diabetic ratswere suffered with gastrointestinal dysfunction with slow transit.2. HO interference by administration of Hemin or ZnPPⅨhad no effecton the body weight or blood glucose of diabetic rats.3. HO interference had no notable effect on HO-2 expression. However,Hemin induced the up-regulation of HO-1 expression, while ZnPPⅨsignificantly inhibited the HO-1 expression.4. The colonic motility of diabetic rats could be influenced by HOinterference. Administration of Hemin might aggravate the decline of colonic motility, while administration of ZnPPⅨmight improve thedeclined colonic motility.5. Administration of ZnPPⅨmight be able to protect ICC by itsblockage of HO-1.6. ICC may produce CO and interve its important role in thegastrointestional motility.7. The alteration of ICC and HO and relationship between them may beimportant etiological factors in the physiology of gastrointestionalmotility, while they may be also one of important mechanisms of whythe declined colonic motility of diabetic rats could be improved oraggravated by the interference of HO.
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