Effect of methalcobalamin on down regulation of insulin-like growth factor-1 gene expression and prevention of diabetic peripheral neuropathy

Jianbo LI; Chengya WANG; Jiawei CHEN; Xiaolu LI; Zhenqing FENG; Hongtai MA

ABSTRACT

BACKGROUND:

Diabetes causes abnormal insulin like growth factor-1 (IGF-1) gene expression, which contributes to initiation and development of peripheral neuropathy.

OBJECTIVE:

To investigate the efficacy of a single dose of methylcabalamin on prevention of experimental diabetic neuropathy and the possible molecular mechanism of its involvement in IGF-1 gene expression.

DESIGN:

Completely randomized and controlled experiment.

SETTING:

Endocrinology Department of the First Affiliated Hospital of Nanjing Medical University.MATERIALS The study was carried out in an Animal Center of Nanjing Medical University. Totally 80 male Sprague Dawley rats (sanitary degree)were randomly selected.

METHODS:

① Totally 64 rats were chosen to be induced diabetic. They were injected intravenously with alloxan dissolved in saline solutions, at the dose of 240 mg/kg. ② Of 16 rats were chosen as normal control group who were injected intravenously with equivalent volume of saline solution. ③ Of 64 established diabetic rats were treated with daily subcutaneous injection of pork regular insulin in combination of protamine zinc insulin (21) then further divided into 2 groups as insulin-treatment diabetic control groups based on different blood glucose levels group 1 with relatively better control of diabetes, group 2 with relatively worse control of diabetes, with 32 rats in each group. Totally 16 rats of each group were treated with methylcobalamin injection intramuscularly with 500 μg/kg body weight, thus correspondingly divided into insulin +methylcobalamin group 1 and insulin+methylcobalamin group 2. The remaining 16 rats of each group as respective insulin-treatment diabetic control groups were treated with equivalent volume of saline. ④ Initiate weight and end weight were measured at beginning of the experiment and after diabetic model was established. Glucose oxidase was used to detect glucose level. 1-deoxy-1-malin was used to detect fructose level. ⑤ Parameters were measured as follows Sensory/motor nerve conduction velocity (SNCV, MNCV) and evoked potential amplitude (EPA) of sciatic nerves detected by evoked electromyogram; IGF-I mRNA by reverse-transcriptase polymerase chain reaction (RT-PCR); IGF-1 peptide by enzyme-linked immunosorbent assay (ELISA). ⑥ One-way analysis of variance was used to analyze the Significance of differences among groups.MAIN OUTCOME

MEASURES:

① Tissue IGF-1 mRNA/ IGF-1 peptide, electrophysiological data of individual groups at different points of the experiment. ② Comparison between individual groups in glucose metabolic parameters and body weights at different points of the experiment.

RESULTS:

Three rats died for diabetic infection or other acute complications and only 77 rats were included in the final statistical analysis.① Body weight and glucose metabolic parameter changes After diabetic model, glucose, fructose level and body weight change between methylcobalamin+insulin treated groups and insulin treated groups were not significant. ② IGF-1 mRNA/peptide changes Tissue IGF-1 mRNA increased significantly in methylcobalamin + insulin treated groups than that in insulin treated groups, respectively (P ＜ 0.05-0.01). Two weeks after diabetic model was established, the sciatic tissue IGF-1 mRNA contents were obviously higher in methylcobalamin + insulin treated group 1 than that in insulin treated group 1 (P ＜ 0.05), but not significantly different from that in NC group; Similarly, tissue IGF-1 mRNA contents were obviously higher in methylcobalamin + insulin treated group 2 than that in insulin treated group 2 (P ＜ 0.05), but lower than that in NC group (P ＜ 0.01); Month 2, tissue IGF-1 contents in methylcobalamin+ insulin treated groups were lower signiiicantly than NC groups, but higher than insulin treated groups (P ＜ 0.05-0.01). By month 3, IGF-1 mRNA level in methylcobalamin+ insulin treated group 2 was not significantly different from that in insulin treated group 2. The IGF-1 peptide levels in nerve tissue changed approximately parallel to IGF-1 mRNA level over time course. ③ Nerve electrophysiological data changes Month 2 and 3, SNCV, MNCV and EPA were significantly higher in methylcobal-amin+ insulin treated group 1 than in insulin treated group 1 (P ＜ 0.05);Month 2, SNCV and EPA were higher in methylcobalamin+ insulin treated group 2 than in insulin treated group 2 (P ＜ 0.05); Month 3, SNCV, MNCV and EPA were significantly lower in methylcobalamin + insulin treated group 2 than in control group (P ＜ 0.05-0.01), whereas no difference was observed between methylcobalamin + insulin treated group 2 and insulin treated group 2.

CONCLUSION:

① Methylcobal has not effect on blood glucose. ②Methylcobal could prevent occurrence of experimental neuropathy through its effect on nerve IGF-1 gene expression of diabetic rats. ③ A better efficacy could be achieved by Methylcobal with a good control of blood glucose level in prevention of diabetic peripheral neuropathy.