| Among diabetes mellitus patients,gastrointestinal dysmotility occurs in up to 25%to 76%of patients with diabetes mellitus.The disorders include gastroparesis,constipation and diarrhea,which all affect the life quality and blood glucose control of the patients.The mechanism hasn't been fully understood.Interstitial cells of Cajal(ICC)are pacemakers of gastrointestinal movement,mediating nerve transmitter production and function.Many studies showed that the depletion of ICC was correlated with many gastrointestinal motility disorders.Stem cell factor(SCF)is the main ligand of c-Kit,a receptor tyrosine kinase and also the surface marker of ICC.SCF-Kit signaling is very important for the maintenance of ICC phenotype,proliferation and differentiation.It is yet controversial about the effect of SCF-Kit signaling on ICC of different ages.In this study,effects of SCF on the pathological changes of colonic ICC of diabetic mice were investigated,and correlation between SCF and depletion of ICC was also discussed.Corresponding research will be helpful to the invention of new effective therapy.Aim: 1.To investigate the pathological change of SCF and ICC in gastrointestinal tract of diabetic mice.2.To investigate the effect of SCF on colonic ICC of diabetic mice.3.To investigate the effect of exogenous SCF on the diabetes-associated depletion of colonic ICC.Methods:1.Establishment of DM mice model,groups and interventions:DM model was established by one single intraperitoneal injection of streptozotocin(STZ)(150mg/kg)in SPF(?)C57/BL6 mice.After modeling,the mice were randomly divided into 4 groups,named group N(normal control)(n=10),DM(diabetic mice without intervention) (n=10),N+anti-SCF(normal mice administered with anti-SCF antibody)(n=10)and DM+SCF(diabetic mice administered with exogenous SCF)(n=l 0).Those in group N and DM were administered with phosphate buffer saline(pH=7.4)quaque die alterna.The administration of anti-SCF(1mg/kg,ip,qod)and SCF(0.2ug/kg,ip,qd) persisted for 6 weeks.The weight and blood glucose of mice were tested.On the third day after the end of intervention,all mice were executed.2.Samples from proximal and distal colon were obtained.Western Blot was used to evaluate the SCF protein level.Serum samples from all mice were obtained.The soluble stem cell factor level in serum was detected by ELISA.3.The quantity of colonic ICC was detected by FACS.The ultrastructure of colonic ICC was observed by transmission electron microscope (TEM).Immunohistochemistry was used to label c-Kit(the surface marker of ICC)positive cells in the mice colon and Western Blot to evaluate the c-Kit protein level.Results:1.①Weight:Mean weights of mice among all groups before treatments showed no statistical difference(P＞0.05).After 6 weeks of treatments, weights were not significantly different between group N and N+anti-SCF(P＞0.05)of the non-diabetic mice,or groups DM and DM+SCF(P＞0.05)of the DM mice.However,those in DM groups were significant lower(P＜0.05)than those in normal groups.②Fasting blood glucose(FBG):The concentrations of FBG in mice of group N and N+anti-SCF were all in the normal range,and those of group DM and DM+SCF were all in the range of the standard DM mice.The latter two groups showed no statistical difference before or after treatments(P＞0.05).2.Colonic SCF:Western Blot showed colonic SCF expression in group DM was significantly lower than that of group N(P＜0.05).SCF expression in group N+anti-SCF was also decreased compared with normal group N(P＜0.05)and not significantly different from that of group DM(P＞0.05).SCF expression in group DM+SCF was higher than group DM(P＜0.05)and not significantly different from group N (P＞0.05).3.Serous SCF:ELISA showed SCF concentration in serum of group DM was significantly lower than that of group N(P＜0.05).Serous SCF in group N+anti-SCF was also decreased compared with normal group N (P＜0.05)and not significantly different from that of group DM (P＞0.05).Serous SCF in group DM+SCF was higher than group DM (P＜0.05)and not significantly different from group N(P＞0.05).4.ICC quantity/c-Kit protein:Compared with group N,FACS showed the mean percentage of ICC of both proximal and distal colon decreased significantly in group DM(P＜0.05)after 6 weeks.Group N+anti-SCF also decreased significantly(P＜0.05)and showed no significant difference from group DM(P＞0.05).However,ICC quatity of Group DM+SCF improved significantly(P＜0.05)and showed no significant difference from group N(P＞0.05).Immunohistochemistry and Western Blot showed c-Kit expression in group DM was significantly lower than that of group N(P＜0.05).c-Kit expression in group N+anti-SCF was also decreased compared with normal group N(P＜0.05).c-Kit expression in group DM+SCF was higher than group DM(P＜0.05),however,it was lower than that of group N(P＜0.05). 5.Ultrastructure of ICC:After 6 weeks of treatments,TEM obsevered colonic ICC in group N displayed the normal morphology,as shown by the intact nuclear membrane structures,full cytoplasmic content,an array of clear and well-developed organelles including mitochondria. They were also shown to tightly connect to neighboring nerve fiber and smooth muscle cells.However,colonic ICC of group DM displayed significant morphological changes,including membrane dissolution, the decrease in the amount of organelles,rough endoplasmic reticulum dilatation,mitochondrial tumefaction,appearance of vacuoles and disappearance of tight junctions among ICC and other cells. Morphological change in ICC of group N+anti-SCF was similar to group DM.Interestingly,treatment with SCF seemed to partially reverse the pathological change of ICC in group DM+SCF.Conclusions:1.The quantitative decline and ultrastructural changes of ICC in the diabetic mice colon may be the pathological factor associated with diabetic dysmotility.2.The decrease of SCF level in serum and colon may lead to the quantitative decline and ultrastructural changes of ICC in the diabetic mice colon.3.Moreover,administration of exogenous SCF partially may reverse these pathological changes of ICC in diabetic mice.
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