Study On The Biological Activity In Pancreatic NIT-1Beta Cells Regulated By NLK And FOXO1and The Relationship Between Two Genes

Objective:Using nandrolone phenylpropionate and LiCl intervened NIT-1cells of pancreatic β cell line under different concentrations of glucose, NLK and FOXO1gene expression were measured in the NIT-1cells. Pancreatic β cell proliferation,insulin secretion and cell cycle were detected and gene expression levels and relationship of the both of two genes were tested. The results will provide a new theoretical basis for the treatment of type2diabetes. Methods:(1) Cultured the NIT-1cells of pancreatic0cell line with four groups of different concentrations of glucose medium:the cells were inoculated in cell culture flask in accordance with5×106/bottle, and then cultured for48hours. In accordance with different concentrations of glucose, divided into four groups:Group1was5.6mmol/L, group2(normal control group) was11.1mmol/L, Group3was16.7mmol/L, Group4was27.6mmol/L, and then cultured for24hours.(2) Intervened with nandrolone phenypropionate and LiCl respectively:In four different concentrations of glucose medium were added10μg/mL nandrolone phenylpropionate and10mmol/L LiCl, to interfere with48hours respectively.(3)Following four methods of experimental monitoring and evaluation:①NIT-1cells survival and growth in different intervention conditions by MTT assay;②Determination of the level of insulin secretion in different intervention conditions measured with radioimmunoassay assay;③NLK and FOXO1protein expression in NIT-1cells were detected by Western-blot technique;④NIT-1cells cycle in the different interventions were tested by PI staining of flow cytometry. Results:(1)The results of NIT-1cells survival and growth by MTT assay:①When the glucose concentration is up to11.1mmol/L, the rate of cell proliferation reach the highest, when the glucose concentrations were over11.1mmo/L, the cell proliferation were descended accompany with glucose concentrations growth.②Cells proliferation levels not significantly higher than the non-intervention group by nandrolone phenylpropionate intervened48hours (P>0.05).③Cells proliferation levels significantly higher than the non-intervention group by LiCl intervened48hours (P<0.05.(2)The results of NIT-1cells insulin secretion levels were measured by radioimmiunoassay:①When the glucose concentration is up to11.1mmol/L, the level of cell insulin secretion reach the highest, when the glucose concentrations were over11.1mmo/L, the cell insulin secretion were descended accompany with glucose concentrations growth.②Application nandrolone phenylpropionate intervened48hours, cells insulin secretion levels not significantly higher than the non-intervention group (P>0.05).③Application LiCl intervened48hours, cell insulin secretion levels significantly higher than the non-intervention group (P<0.05).(3)The results of NIT-1intracellular NLK and FOXO1protein expression levels which were measured by protein blotting techniques in different interventions:①Application nandrolone phenylpropinate intervened NIT-1cells under the different concentration of glucose48hours, the NLK protein expression levels increased significantly than the non-intervention group (P<0.05).②Application LiCl intervened NIT-1cells under the different concentration of glucose48hours, the NLK protein expression levels increased than the non-intervention group (P<0.05)③Application nandroline phenylpropionate and LiCl actived the NLK gene expression48hours, FOXO1protein expression level was reduced with increasing NLK protein expression level (P<0.05).(4)The results of the changes of the cell cycle which were measured by PI staining of flow cytometry in different interventions:①When the glucose concentration is up to11.1mmol/L, the percentage of cells in the S phase, G2/M phase and PI of the cell cyle reach the highest, when the glucose concentrations were over11.1mmo/L, the percentage of cells in the S phase, G2/M phase and PI of the cell cyle were descended accompany with glucose concentrations growth (P<0.05).②Application nandrolone phenylpropionate intervened NIT-1cells under the different concentration of glucose48hours, the percentage of cells in the S phase, G2/M phase and PI of the cell cyle with no drug intervention group compared not chandged significantly (P>0.05);③Application LiCl intervened NIT-1cells under the different concentration of glucose48hours, the percentage of cells in the S phase, G2/M phase and PI of the cell cyle compared with no drug intervention group increased significantly (P<0.05). Conclusions:(1)Nandrolone phenylpropionate could promote NLK gene expression, thereby inhibit the FOXO1gene expression, but the increase of cell proliferation and insulin secretion levels is not significantly.(2)LiCl could promote NLK gene expression, thereby inhibit the FOXO1gene expression and then promote NIT-1cells proliferation and insulin secretion; LiCl promotes cell proliferation by cell cycle arrest in the S phase and G2/M phase.(3)In NTT-1cells of pancreatic0cell line, FOXO1protein levels was reduced with increasing NLK protein expression levels.