| Diabetic gastroenteropathy mainly including Diabetic gastroparesis (DGP) andcolonic dysfunction, occurs frequently in patients with diabetes mellitus(DM), whichaffects the life quality and blood glucose control of the patients. However, themechanism hasn’t been fully understood. In human and animal studies the smoothmuscle cells (SMCs) in diabetic gastroenteropathy appeared atrophy, fibrosis, evenapoptosis and necrosis detectable by histology, accompany with the significantreduction of the SMCs constraction and its related protein production. In fact, thesedisorders are multifactorial and related with systemic autonomic and enteric nervoussystems, interstitial cells of Cajal (ICC), SMCs, hyperglycemia and trophic factors.MAPK-ERK and PI3K-AKT signaling were two important pathways regulating theSMCs proliferation in gastroenterology. Mitochondrial apoptosis pathway regulatingby bcl-2family was an important way to regulate early apoptosis of the cells. It hasbeen proven that diabetic gastroenteropathies should be considered a form ofgastrointestinal neuromuscular dystrophy rather than a “functional” disorder. In recentyears, loss of trophic support has received more attention as a mediator ofdiabetes-associated cellular degeneration or injury. Particularly important in thisregard was insulin-like growth factor (IGF-1), which played an important role in thedevelopment, differentiation, proliferation process of SMCs in the gastrointestinaltract. Our previous experiments confirmed that IGF-1induced gastric and colonicSMCs to produce SCF, and had a protective effect with ICC. Some findings stronglysupported the notion that reduced IGF-I singnaling in diabetes was an independent factor of this disease. Chronic high glucose was toxic to SMCs, impairing cellularfunction as observed in DGP. However, the mechanisms underlying SMCsdysfunction are still unclear. It is not unclear that whether high glucose affects theexpression of IGF-I and SCF. In this study, we tested the hypothesis that high glucoseconcentrations adversely affect survival of cultured rat SMCs through the modulationof the related proliferaion and apoptosis singnaling pathway.Aim:1. To indentify that wheather high glucose can affect the proliferation and apoptosisof colonic SMCs and the related molecular mechanism of cellular injury.2. To investigate the effects of high glucose on the expression of endogenousinsulin-like growth factor-1(IGF-1) and stem cell factor (SCF) in colonic SMCs.Methods:1. Isolation, culture and identification of colonic SMCs: SD rat was anesthetizedby intraperitoneal injection of10percentage chloral hydrate. SMCs were isolatedand cultured from the colon by enzymolysis. The colonic SMCs were culturedand subcultured in DMEM supplemented with10%fetal bovine serum. The cellswere identified by SMα-actin immunofluorescence methods.2. The colonic SMCs were devided into three groups, normal glucose group(5.5mmol/L glucose), hyperosmolar control group (5.5mmol/L glucose plus19.5mmol/L mannitol) and high glucose group (25mmol/L glucose).(1) We used CellCounting Kit-8to analyze the proliferation of SMCs in different time (12,24,48,72,96hours); we analyzed the cell cycles of SMCs at24hours by flow cytometry.(2) The colonic SMCs were treated with high glucose (25mmol/L glucose) atdifferent times (0,5,15,30,45,60min), the phosphorylation level of ERK1/2,AKT in these cells were measured by Western bloting; the colonic SMCs weretreated with the above-mentioned three groups, the phosphorylation level of ERK,AKT in these cells was measured by Western bloting at30minutes.3. The colonic SMCs were devided into the obove-mentioned three groups, we analyzed apoptosis of SMCs at72hours by flow cytometry. The expression ofbax、bad and bcl-2in these cells was measured by Western bloting at72hours.4. The rat were devided into two groups, the STZ-diabetic group and the controlgroup, Real time quantitive-PCR and Western blotting were performed to analyzethe mRNA and protein expression of IGF-1in colon tissue of rats.5. The colonic SMCs were devided into the obove-mentioned three groups, ELISAwas designed to measure rat IGF-I in SMCs culture supernates. Real timequantitive-PCR and Western blotting were performed to analyze the mRNA andprotein expression of IGF-1in SMCs.6. The colonic SMCs were devided into the obove-mentioned three groups andexogenous IGF-1group (25mmol/L glucose plus100ug/L IGF-1). The expressionof SCF in these cells was measured by Western bloting and Real timequantitive-PCR.Results:1. Culture and identification of colonic SMCs: In primary culture, gastric and colonicSMCs were attached to the culture vessels by48h, triangle-shaped orspindle-shaped with centrally located nuclei. SMCs were proliferated3-5d later,and reached confluence after7to10days with a “hill-and-valley” pattern. Greenimmunofluorescence was found in cytoplasm of most cultured cells which denotedpositive reaction. The nuclei showed blue which was treated Hoechst.2. The effect of different concentrations of glucose on proliferation of colonic SMCs:Compared with normal glucose, high glucose treatment inhibited the proliferationof rat colonic SMCs (P＜0.05). High glucose treatment caused SMCsaccumulation in the G1phase (P＜0.05)with a significant decrease in the numberof cells in the S phase (P＜0.05). High glucose treatment down-regulated thephosphorylation level of AKT(P＜0.05). There was no significant difference withthe phosphorylation level of ERK.3. The effect of different concentrations of glucose on apoptosis of colonic SMCs: High glucose treatment induced the lower survival rate of colonic SMCs (P＜0.05),and higher ealy apotosis rate (P＜0.05). High glucose treatment up-regulated theexpression of pro-apoptosic protein bax and bad (P＜0.05), and down-regulated theexpression of anti-apoptosic protein bcl-2(P＜0.05).4. The expression of IGF-1and SCF in the colonic tissue of diabetic rat: Theexpression of IGF-1and SCF were significantly decreased in the colonic tissue ofdiabetic rat (P＜0.05).5. The effect of different concentrations of glucose on the expression of endogenousIGF-1: High glucose treatment decreased the content of IGF-I in SMCs culturesupernates(P＜0.05), and the expression of IGF-I mRNA and protein(P＜0.05).6. The effect of different concentrations of glucose on the expression of endogenousSCF: High glucose treatment decreased the expression of IGF-I mRNA andprotein(P＜0.05), While exogenous IGF-1could increase the expression of SCFmRNA induced in high glucose environment.Conclusions:1. High glucose might inhibite the proliferation of rat colonic SMCs bydown-regulating the phosphorylation level of AKT.2. High glucose might induce the apoptosis of rat colonic SMCs byActivating the mitochondrial apoptosis pathway.3. High glucose decreased the expression of endogenous IGF-1and SCF in SMCs, butexogenous IGF-1could reverse the reduction of the expression of SCF in highglucose environment. |
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