| Gastrointestinal dysmotility is one of diabetic complications. Cell apoptosis plays a critical role in diabetes mellitus and its complications. The PI3K/Akt signal transduction pathway which is made up of phosphatidylinositol 3-kinase and Akt is associated with glucose metabolism and apoptosis of diabetes mellitus. The study aims to explore the relationship between diabetic colon dysmotility and apoptosis of colonic smooth muscle cell(SMC) and interstitial cell of Cajal(ICC). We also investigate the intervention effects and pathgogenesis of insulin and insulin-like growth factor-1 (IGF-1) to provide theoretical evidences for pathgogenesis and therapy of diabetic colon complication.Part?The Study on Apoptosis of Colonic SMC and ICC in Diabetic Colon DysmotilityObjective:To investigate the apoptosis of colonic SMC and ICC and the protein expression of connexin 43 (Cx43) of ICC in diabetic colon dysmotility.Methods:Thirty-six male SD rats were randomly divided into 4 groups which included normal 6w group,normal 10w group,diabetes mellitus (DM) 6w group and DM 10w group (n=9). Diabetes mellitus was induced by streptozotocin. Weight, fasting blood glucose (FBG) and gastrointestinal transit rate were detected, and the changes in the smooth muscle of colon were examined by HE staining. Real-time fluorescence quantitative PCR and Western Blot were performed to analyze the mRNA and protein expression levels of bax, bcl-2 and caspase-3 of colonic SMC. TUNEL was used to detect apoptosis index (AI) of colonic SMC and ICC. Immunohistochemistry was used to detect the protein expression of Cx43 of ICC.Results:(1)FBG of DM group was higher than that of normal group. Weight and gastrointestinal transit rate of DM group were lower than those of normal group. The smooth muscle of colon in DM group became thinner than that in normal group. The mRNA and protein expression levels of bax and caspase-3 in DM group were higher than those in normal group and the mRNA and protein expression levels of bcl-2 in DM group were lower than those in normal group. AI of colonic SMC in DM group was higher than that in normal group. The protein expression of Cx43 of ICC in DM group was lower than that in normal group, and there were significant differences(P<0.05).(2)FBG of DM 10w group was higher than that of DM 6w group. Gastrointestinal transit rate of DM 10w group was lower than that of DM 6w group. The smooth muscle of colon in DM 10w group became thinner than that in DM 6w group. The mRNA and protein expression levels of bax and caspase-3 in DM 10w group were higher than those in DM 6w group and the mRNA and protein expression levels of bcl-2 in DM lOw group were lower than those in DM 6w group. AI of colonic SMC in DM 10w group was higher than that in DM 6w group. The protein expression of Cx43 of ICC in DM 10w group was lower than that in DM 6w group, and there were significant differences(P<0.05).(3)There was no difference between AI of colonic ICC in DM group and that in normal group (P>0.05), and so was between DM 10w group and DM 6w group. Conclusion:Increasing apoptosis of colonic SMC,decreasing colonic ICC and less Cx43 expression of colonic ICC maybe one of mechanisms of diabetic colon dysmotility, and the changes above became more significant with disease developing. Decreasing colonic ICC maybe not associated with the apoptosis of colonic ICC.Part?The Study on Apoptosis Pathway of Coionic SMC in Diabetic Colon DysmotilityObjective:To explore the apoptosis pathway of colonic SMC in diabetic colon dysmotility. Methods:There were totally 2 groups:normal 10w group and DM 10w group(n=9). Immunohistochemistry was used to detect the protein expressions of CASPASE-9, CASPASE-12 and CASPASE-8 of colonic SMC. Results:The CASPASE-9 expression of colonic SMC in DM 10w group was higher than that in normal 10w group, and there was significant difference(P<0.01). There was no significant difference between the expressions of CASPASE-12 and CASPASE-8 of colonic SMC in DM lOw group and those in normal 10w group (P>0.05). Conclusion:The apoptosis of colonic SMC in diabetic colon dysmotility maybe associated with the mitochondrial pathway, and not associated with the pathway induced by endoplasmic reticulum and death receptor. Part?The Effects of Insulin and IGF-1 on Apoptosis of Colonic SMC in Diabetic Colon DysmotilityObjective:To explore the effects of insulin and IGF-1 on apoptosis of colonic SMC in diabetic colon dysmotility. Methods:One hundred and seventeen male SD rats were randomly divided into 13 groups which included normal 6w group, normal lOw group, DM 6w group, DM lOw group, IGF-16w group, IGF-1 l0w group, Ins 6w group and Ins lOw group(including high-dosage, moderate-dosage and low-dosage)(n=9). Weight, FBQ gastrointestinal transit rate and serum levels of insulin and IGF-1 were detected, and the changes in the smooth muscle of colon were examined by HE staining. Real-time fluorescence quantitative PCR and Western Blot were performed to analyze the mRNA and protein expression levels of bax, bcl-2 and caspase-3 of colonic SMC. TUNEL was used to detect AI of colonic SMC. Results: (1)Compared with DM 6w group, all insulin and IGF-16w groups down-regulated the mRNA and protein expression levels of bax and caspase-3, up-regulated the mRNA and protein expression levels of bcl-2, decreased AI and increased gastrointestinal transit rate, and there were significant differences(P< 0.05). The effects of high-dosage insulin were better than those of moderate-dosage insulin and low-dosage insulin, and there were significant differences(P<0.05). There was no significant difference between moderate-dosage insulin and low-dosage insulin(P> 0.05).(2)Compared with DM lOw group, all insulin and IGF-1 lOw groups down-regulated the mRNA and protein expression levels of bax and caspase-3, up-regulated the mRNA and protein expression levels of bcl-2, decreased AI and increased gastrointestinal transit rate, and there were significant differences(P<0.05). The effects of early high-dosage insulin were better than those of moderate-dosage insulin, low-dosage insulin and advanced high-dosage insulin, and there were significant differences(P< 0.05). There was no significant difference among moderate-dosage insulin, low-dosage insulin and advanced high-dosage insulin(P> 0.05). (3)Compared with DM groups, all insulin groups decreased FBG and increased serum level of insulin, and there were significant differences(P<0.05). The serum level of insulin was significantly inversely correlated with AI of colonic SMC(r=-0.709, P?.01). All IGF-1 groups increased serum level of IGF-1, and there were significant differences(P< 0.01). The serum level of IGF-1 was significantly inversely correlated with AI of colonic SMC(r=-0.857, P<0.01). All IGF-1 groups did not decrease FBG. Conclusion:Both insulin and IGF-1 can improve the apoptosis of colonic SMC in diabetic colon dysmotility, and the effects of early high-dosage insulin were better than those of moderate-dosage insulin, low-dosage insulin and advanced high-dosage insulin.PartIV The Effects of High Glucose, Insulin and IGF-1 on PI3K/Akt Pathway of Colonic SMCObjective:To explore the effects of high glucose, insulin and IGF-1 on proliferation, apoptosis and PI3K/Akt pathway of colonic SMC. Methods:In vitro culture of the rat colonic SMC is performed by the means of tissue sticking. The colonic SMC were devided into ten groups including normal control group, high glucose group(33.3 mmol/L glucose), high glucose+LY10 group(33.3 mmol/L giucose+10?mol/L LY294002), mannitol group(33.3 mmol/L mannitol), Ins group(100 nmol/ L insulin), Ins+LY10 group(100 nmol/L insulin+10?mol/L LY294002), Ins+LY50 group(100 nmol/L insulin+50?mol/L LY294002), IGF-1 group(100?g/L IGF-1), IGF-1+LY10 group(100?g/LIGF-1+10?mol/L LY294002), IGF-1+LY50 group(100?g/L IGF-1+50 umol/L LY294002). We used MTT to analyze the proliferation of SMC and flow cytometry to analyze the apoptosis. Western Blot was performed to analyze the protein expression levels of Akt, p-AktThr308, p-AktSer473 and CASPASE-9 of colonic SMC. Results:(1)Sufficient rat colonic SMC were obtained by culture in vitro.(2)Compared with normal control group, the proliferation was decreased and apoptosis was enhanced in high glucose group and high glucose+LY10 group, and there were significant differences(P<0.01). The changes above became more significant in high glucose+LY10 group, and there were significant difference compared with high glucose group (P<0.01). The expression of p-AktThr308 in high glucose group was lower than that in normal control group, and there was significant difference(P<0.01). Compared with normal control group, the expressions of p-AktSer473 and Akt in high glucose group showed no significant difference (P>0.05). The proliferation, apoptosis and expressions of p-AktThr308, p-AktSer473 and Akt in mannitol group showed no significant difference (P>0.05).(3)Compared with normal control group, more proliferation, less apoptosis, more expression of p-AktSer473 and less expression of CASPASE-9 were observed in Ins group, and there were significant differences(P<0.01). Less proliferation, more apoptosis, less expression of p-AktSer 73 and more expression of CASPASE-9 were observed in Ins+LY10 group and Ins+LY50 group, and there were significant differences(P<0.01). The changes above became more significant in Ins+LY50 group than those in Ins+LY10 group, and there were significant differences(P<0.01). The expressions of p-AktThr308 and Akt showed no significant difference among normal control group, Ins group, Ins+LY10 group and Ins+LY50 group(P>0.05).(4) Compared with normal control group, more proliferation, less apoptosis, more expression of p-AktSer473 and less expression of CASPASE-9 were observed in IGF-1 group, and there were significant differences(P<0.01). Less proliferation, more apoptosis, less expression of p-AktSer473 and more expression of CASPASE-9 were observed in IGF-1+LY10 group and IGF-1+LY50 group, and there were significant differences(P<0.01). The changes above became more significant in Ins+LY50 group than those in IGF-1+LY10 group, and there were significant differences(P< 0.01). The expressions of p-AktThr308 and Akt showed no significant difference among normal control group, IGF-1 group, IGF-1+LY10 group and IGF-1+LY50 group(P> 0.05).Conclusion:We concluded that high glucose may lead less proliferation and more apoptosis of colonic SMC by P13K/Akt pathway and the results were not associated with osmotic pressure. Insulin and IGF-1 may lead more proliferation and less apoptosis of colonic SMC by P13K/Akt pathway. |
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