| Disorder of gastrointestinal (GI) motility is one of major GI diseases. Gastrointestinal dysmotility includes Diabetic gastroparesis (DGP) and colonic dysfunction, which affects the life quality and blood glucose control of the patients, and which mechanism hasn't been fully understood. In the patients with diabetic GI dysmotility, a decrease in ICC number and cellular degradation of ICC were reported to be widely spread and observed in the stomach and colon. SCF-Kit signaling has been shown to be important for the maintenance of ICC phenotypes, proliferation and differentiation. Reduced SCF was reported in the stomach and colon in the diabetic mice, and exogenous SCF partially reverses the pathological changes of ICC in diabetic mice. 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. In this study, we investigated whether exogenous IGF-1 regulated synthesis of SCF in gastrointestinal SMC and its related mechanism.Aim:1. To explore the effect of insulin-like growth factor 1 (IGF-1) on expression of stem cell factor (SCF) in rat gastric and colonic smooth muscle cell.2. To explore the effect of anti-IGF-1Rαantibody on expression of SCF in rat gastric and colonic smooth muscle cell.3. To explore the molecular mechanism of SCF upregulation in rat gastric and 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. Treated with increasing concentrations (0, 5, 10, 50, 100, 150μg/L) of IGF-1 for 16h, or with 100μg/L IGF-1 at different times (0, 8, 16, 24, 48h), the rat gastric and colonic SMC were routinely cultured. The expression of SCF in these cells was measured by Western blot and quantitative Reverse Transcription-Polymerase Chain Reaction.3. The rat gastric and colonic SMC were incubated with IGF-1 (100μg/L) and anti-IGF-1Rαantibody (0, 50, 100, 150μg/L) at different concentrations respectively for 16 hours. The expression of SCF in these cells was measured by Western blot and quantitative Reverse Transcription-Polymerase Chain Reaction.4. The rat gastric and 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. 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 gastric and colonic SMC: In primary culture, gastric and colonic SMC were attached to the culture vessels by 24h, triangle-shaped or spindle-shaped with centrally located nuclei. SMC were proliferated 3-5d later, and reached confluence after 14 days with a"hill-and-valley"pattern. Red immunofluorescence was found in cytoplasm of most cultured cells which denoted positive reaction. The nuclei showed blue which was treated Hoechst.2. The effect of different concentrations of IGF-1 on expression of SCF in rat gastric and colonic smooth muscle cell: Very low level of SCF was expressed in gastric and colonic SMC cultured in the bovine serum free medium. IGF-1 in low concentration (5 and 10μg/L) had no effect on the expression of SCF in SMC (P>0.05), and the expression of SCF mRNA and protein were increased with IGF-1 in higher concentration (50, 100, 150μg/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.3. The effect of different times of IGF-1 on expression of SCF in rat gastric and 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 was at the 16th hour in cultured with IGF-1 (P<0.05). There was no significant difference between 0h and 48h treated with IGF-1 (P>0.05).4. The effect of different concentrations of anti-IGF-1Rαantibody on expression of SCF in rat gastric and colonic smooth muscle cell: After gastric and colonic SMC were incubated with IGF-1 (100μg/L) and anti-IGF-1Rαantibody, the anti-IGF-1Rαantibody can inhibit the expression of SCF in SMC in a dose-dependent manner, and there was significant difference between the adjoining groups (P<0.05).5. The effect of PD-98059 and LY-294002 on expression of the IGF-1-induced SCF in rat gastric and 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. 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 with IGF-1.2. Anti-IGF-1Rαantibody can inhibit the expression of SCF in SMC in a dose-dependent manner.3. IGF-1 induces SCF via an ERKMAPK signal transduction pathway in gastric and colonic SMC.
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