Effect Of Blood Glucose And Insulin On Interstitial Cells Of Cajal In Diabetic Mice Colon

Gastrointestinal dysmotility is a frequent complication of diabetes mellitus(DM).As pacemaker of the gastrointestinal contract activities and mediator of enteric nervous system to the gastrointestinal smooth muscles,interstitial cells of Cajal(ICC)plays an important role in this process and has attracted much attention in the field.Decrease in cell numbers and change in the ultra-structures of ICC in the stomach and the colon of diabetic rats were recently reported,and they were also shown to be associated with Gastrointestinal dysmotility in DM.There are many problems without clarification on the mechanism of ICC changes in DM. DM is characterized by chronic hyperglycemia as well as insulin deficiency or resistance.In consistent with previous studies, hyperglycemia caused ICC lesion,but it was recently reported that hyperglycemia could help in maintaining ICC quantit.Both smooth muscle cells and intestinal neurons express insulin receptors and the natural ligand of SCF.The SCF-Kit system plays an important role in proliferation,development and phenotype maintenance of ICC,insulin deficiency may play an important role in ICC lesion in DM.Investigate the effect of insulin and blood glucose on ICC in the DM mouse model will be helpful to the invention of new efficacious remedies to prevent ICC lesion in DM.Aim:1.To establish Mouse DM model.2.To study the changes of cell quantities and the ultrastructure of ICC in the colon of diabetic mice.3.To investigate changes of ICC in the colon of normal and DM mice in response to hyperglycemia.4.To investigate the effect of insulin on ICC in DM mice colon.Methods:1.DM mouse model was established by intraperitoneal injection of streptozotocin(STZ)(150mg/kg);the control group was injected with the same dose of citrate buffer solution.2.Mice were divided into 5 groups:group N(normal control mice, n=10),DM(diabetic mice without treatment,n=10),DM+Ins(diabetic mice administered with insulin,n=10),DM+Ins+Glu(diabetic mice treated with insulin and high glucose,n=10),and N+Glu(normal mice administered with high glucose,n=10).3.Mice were recorded for their glucose and raised for total 6 weeks.All mice were sacrificed three days after completion of the treatments,at that time,bloods were drawn and insulin levers were detected by ELISA. 4.Samples from colonic tissues were obtained in each groups respectively,the pencent of c-kit positive cells were detected by Flow cytometry(FCM);the distribution of ICC was observed by immunohistochemistry(IHC)and for each tissue section,5 randomly selected fields were counted for ICC under high-power microscope.5.Samples from colonic tissues were obtained in each groups respectively,the ultrastructure of ICC was observed with transmission electron microscopy(TEM).Results:1.Compared with the control group(group N),the blood glucose in DM mice(group DM)was increased(p＜0.05);however,when administrated with insulin(group DM+Ins),their blood glucose level were remained similar with group N(p＞0.05);when DM mice administrated with the same dose of insulin as group DM+Ins,then administrated with glucose (group DM+Ins+Glu),their blood glucose level were between group N and DM(p＜0.05);comparison with group N,there was a transient hyperglycemia in normal mice after injected glucose(group N+Glu) (p＜0.05).2.Serum insulin levels were similar among group DM,DM+Ins and DM+Ins+Glu(p＞0.05),and between groups N and N+Glu(p＞0.05),but those in all DM groups(DM,DM+Ins and DM+Ins+Glu)were significantly deceased(p＜0.05).3.Detected the pencent of c-kit positive cells in mice colon by FCM,we found:comparison with the group N,ICC quantities decreased in colons of mice from all other groups(DM,DM+Ins,DM+Ins+Glu and N+Glu); the percentages of ICC were similar in group DM and DM+Ins(P＞0.05), but both of which were significantly lower than that of DM+Ins+Glu (P＜0.05).4.ICC were mainly localized between ring and longitudinal muscles of the colon.The quantities of ICC in mice colon detected by IHC further confirmed the results obtained by FCM.5.The characters of ICC in the colon of control group integrated basal lamina,abundant mitochondrion,heterochromatism distributed along nuclear membrane was the major.Dissolved cytoplasm,damaged organelles and formation of vacuoles were found in the colon of DM mice.Ultra-structure of ICC abnormal change in group DM and N+Glu were most remarkable.Conclusions:1.Blood glucose increase was company with insulin lever decline in DM mice.2.Mice with DM displayed the decline in ICC quantity and pathological changes in ICC ultra-structures.3.Both transient and persistent hyperglycemia caused severe damages to ICC ultra-structures and decline in ICC quantity.4.While certain degree of hyperglycemia in DM mice could partially prevent ICC quantitative decline. 5.Endogenous insulin deficiency induced ICC quantitative declines and had no direct effect on ICC ultra-structures.6.Endogenous insulin deficiency induced ICC quantitative declines that could not be restored by addition of exogenous insulin.Exogenous insulin restored the ICC ultra-structure,possibly by controlling hyperglycemia.