| Diabetic gastrointestinal (GI) dismotility is a metabolic diseases affects the gastrointestinal nerve (autonomic nervous system, gastrointestinal myenteric plexus) and muscle, and the specific mechanism is not clear. Study confirmed that the number and structural abnormalities of interstitial cells of Cajal (ICC) in gastrointestinal is one of the reasons of diabetic GI dismotility; Stem cell factor (SCF) plays an important role in the gastrointestinal tract development, differentiation, proliferation process of ICC, SCF reduced in the gastrointestinal tract in diabetic, and exogenous SCF can improve or reverse the disease of ICC and gastrointestinal motility disorder; Insulin-like growth factor 1 (IGF-1) is able to prevent from depletion of ICC, and which is the essential for ICC existence. So we presumed that SCF expression in gastrointestinal SMC was stimulated by IGF-1. Early experiments confirmed that IGF-1 induced gastric and colonic SMCs of normal rat produce SCF by ERKMAPK signaling pathway on the cellular level, and this study further confirmed the mechanism in genetic level; In addition, the gastrointestinal SMCs were all from normal rat in early experiments, and it did not reflect the real disease status of the gastrointestinal in DM. This experiment established the DM model to explore the effect and molecular mechanism of IGF-1 on the expression of SCF which produced by SMCs in cellular level. Aim:1. To explore the effect of insulin-like growth factor 1 (IGF-1) on expression of stem cell factor (SCF) in rat colonic smooth muscle cell which was transfected with ERK siRNA plasmid.2. To explore the effect of insulin-like growth factor 1 (IGF-1) on expression of stem cell factor (SCF) in normal and DM rat colonic smooth muscle cell.3. To explore the molecular mechanism of SCF upregulation in normal and DM rat colonic smooth muscle cell treated with IGF-1.Methods:1. Isolation, culture and identification of gastric and colonic SMC: SD rat was sacrificed by breaking down cervical vertebrae. SMC were isolated and cultured from the gastric antrum and colon by enzymolysis. Gastrointestinal SMC were cultured and subcultured in DMEM supplemented with 10% fetal bovine serum. The cells were identified byα-actin immunofluorescence methods.2. Rat colonic SMC were treated by IGF-1 with different times (0, 5, 15, 30, 45, 60min) and different concentrations (0, 50, 100, 150μg//L), in order to get the peak time and the best consentration of phosphorylated ERK1/2 by western-blot. Rat colonic SMC were transfacted by ERK1/2 siRNA plasmid to get the expression of ERK1/2 and SCF which induced by IGF-1. The expression of p-ERK, t-ERK and SCF in these cells was measured by Western blot and quantitative Reverse Transcription-Polymerase Chain Reaction.3. Treated with increasing concentrations (0, 5, 10, 50, 100, 150μg/L) of IGF-1 for 16h in normal rat colonic and 24h in DM rat colonic, or with 100μg/L IGF-1 at different times (0, 8, 16, 24, 48h), the rat colonic SMC were routinely cultured. The expression of SCF in these cells was measured by Western blot and quantitative Reverse Transcription-Polymerase Chain Reaction. 4. The rat colonic SMC were pretreated with specific mitogen-activated protein (MAP) kinase kinase (MEK1) inhibitor PD-98059 and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 for 1h then cultured with IGF-1 (100μg/L) for 16h in normal rat colonic and 24h in DM rat colonic. The expression of SCF in these cells was measured by Western blot and quantitative Reverse Transcription-Polymerase Chain Reaction.Results:1. Culture and identification of colonic SMC: In primary culture, gastric and colonic SMC were attached to the culture vessels by 24h, transparent cytoplasm, uniform density, triangle-shaped or spindle-shaped with centrally located nuclei. The cells were arranged in parallel around on th 5th day, part of the SMC multi-layer overlap, some single, synaptic connectioned between cells with a"hill-and-valley"pattern. SMC were about to merge into pieces and into a multi-layer cross each other in 7 days, then they could be passaged. But in which, the DM rats colonic SMC needed 48h to attach to the culture vessels, the morphology was similar to the normal colonic SMC, but it grew slowly, the SMC of DM rat began to proliferate on the 5th day, the growth in the 10th day was similar to the normal in the 5th, and that in the 14th was similar to the normal in the 7th. Red immunofluorescence was found in cytoplasm of most cultured cells which denoted positive reaction. The nuclei showed blue which was treated Hoechst.2. The peak time of phosphorylated ERK1/2 expression in colon SMC was about 15min which induced by IGF-1(p<0.05), and the best concentration of IGF-1 was 100μg/L(p<0.05). The expression of phosphorylated ERK1/2 and SCF in colonic SMC which was transtacted by ERK1/2 siRNA plasmid and then induced by IGF-1, was lower than that in control group(p<0.05)3. The effect of different concentrations of IGF-1 on expression of SCF in normal and DM rat colonic smooth muscle cell: Very low level of SCF was expressed in colonic SMC cultured in the bovine serum free medium.The expression of SCF mRNA and protein were increased with IGF-1 in 50μg/L(P<0.05), and in which, IGF-1 in 100μg/L may be the effective final concentration in vitro. IGF-1 in 150μg/L got similar result with IGF-1 in 100μg/L.The effect of DM rat colonic is similar to the normal rat.4. The effect of different times of IGF-1 on expression of SCF in normal and DM rat colonic smooth muscle cell: SCF protein and mRNA expression gradually increased in a time-dependent manner after treated with IGF-1 in 100μg/L, and the peak of SCF expression in normal rat colonic was at the 16th hour and the DM rat colonic was the 24th in cultured with IGF-1 (P<0.05).5. The effect of PD-98059 and LY-294002 on expression of the IGF-1-induced SCF in normal and DM rat colonic smooth muscle cell: Treatment of gastric and colonic SMC with specific inhibitor of MEK1 (PD-98059) significantly suppressed IGF-1 induced SCF expression (P<0.05). It was little effect of LY-294002 on the expression of SCF (P>0.05).Conclusions:1. The expression of phosphorylated ERK1/2 was increased in colonic SMC which induced with IGF-1, and the expression of phosphorylated ERK1/2 and SCF was decreased after transfacted ERK1/2 siRNA plasmid in colonic SMC. It confirmed that SCF from colonic SMC induced by IGF-1 may be invoved through the ERKMAPK pathway.2. IGF-1 can induce SCF expression in gastric and colonic SMC in both dose- and time-dependent manners. IGF-1 in 100μg/L may be the effective final concentration, and the peak of SCF expression was at the 16th hour in normal rat colonic and 24 hour in DM rat colonic with IGF-1. 3. IGF-1 induces SCF via an ERKMAPK signal transduction pathway in gastric and colonic SMC. |
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