| Objective: 1.To observe the expression of insulin-like growth factor-1(IGF-1) in rat schwann cells(RSCs) and apoptosis of RSCs under the condition of various levels of high glucose. 2.To observe whether exogenous IGF-1 can protect RSCs from apoptosis under high glucose via neuritin expression and a possible cellular signal mechanism. Methods: 1.We detected the IGF-1’s expression using RT-QPCR in RSCs cultured in various levels of glucose(5.6 mM,25 mM,50 mM,100 mM,125 mM, and150 mM GS) for 48 h. 2.We examined the anti-apoptotic effect of various levels of exogenous IGF- 1 on RSCs cultured in high glucose for 36h: IGF- 1 treatment group 1(125 mM GS + 0.1 ng/mL IGF- 1), IGF- 1 treatment group 2(125 mM GS + 1ng/mL IGF- 1), IGF- 1 treatment group 3(125 mM GS + 10ng/mL IGF- 1), and IGF- 1 treatment group 4(125 mM GS + 100ng/mL IGF- 1), in contrast to non-IGF- 1 treatment control group(125 mM GS). Apoptosis rate was assayed using flow cytometry and neuritin expression of RSCs was assayed using RT-QPCR or Western blot, respectively. 3.We observed the apoptosis of neuritin-silenccd RSCs treated with 50μg/mL BAF and/or 10ng/ml IGF-1 for 24 h using Annetin Ⅴ-PE /7-ADD. The cells were divided into following different groups: the control group, the non-virus group, and the neuritin-silenced group. IGF- 1 treatment neuritin-silenced RSCs group 1, BAF treatment neuritin-silenced RSCs group 2, IGF-1 and BAF treatment neuritin-silenced RSCs group 3. 4.We observed the apoptosis of neuritin-over-expressed RSCs in high glucose(125mM), treated with 10μg/mL LY294002 and/or 10ng/ml IGF-1 for 36 h using Annetin Ⅴ-PE /7-ADD. The cells were divided into different groups: control group, non-virus group, neuritin-over-expressed RSCs group, IGF- 1 treatment RSCs group 1, IGF-1 and LY294002 treatment RSCs group 2, IGF-1 treatment neuritin-over-expressed RSC group 3, and IGF-1 and LY294002 treatment neuritin-over-expressed RSCs group 4. Results: 1.IGF-1 mRNA was expressed in RSCs. There was no significant difference among groups in different levels of glucose(p>0.05). 2.Compared with control group, the mRNA and protein expression of neuritin gene and cell apoptosis rate in both IGF- 1 treatment group 3 and IGF- 1 treatment group 4 significantly increased, respectively(p<0.05). Between the group 3 and 4, there was not significantly different(p > 0.05). 3.The effect of BAF treatment on survival of RSCs with silenccd neuritin was observed. Compared with neuritin-silenced RSCs group and group 1, the cell apoptosis rate in both group 2 and group 3 were significantly decreased, respectively(p<0.05), whereas group 1 had similar cell apoptosis rates(p>0.05). But between group 2 and 3, there was no significant difference(p>0.05). It showed that exogenous IGF-1 alone does not reduce the apoptosis of the neuritin-silenced RSCs whereas BAF alone or with IGF-1 lowers the apoptosis. 4 The effect of LY294002 treatment on survival of RSCs with neuritin-over-expressed under high glucose was observed. Compared with control group, the cell apoptosis rates in group 1 and group 3 or group 4 were significantly decreased, whereas group 2 had increased rates. Compared with group 1,the cell apoptosis rate in group 2 had increased(p<0.05). But between group 3 and 4, there was no significant difference(p>0.05). It suggests that LY294002 does not block the protection of IGF-1 against the neuritin-over-expressed RSCs apoptosis under high glucose. Conclusion: 1.RSCs secretes IGF-1,which is not greatly affected by high glucose. 2.IGF-1 partially prevents apoptosis of RSCs from occurring under the high glucose conditions. However, it dose not prevent apoptosis of neuritin-silenced RSCs. In addition, IGF- 1 prevents, to some extent, the reduction of the mRNA and protein expression of neuritin gene in RSCs exposed to high glucose in vitro in this study. In this case, neuritin may be located downstream of IGF-1 and may mediate some of the effects of IGF-1. 3.BAF, a inhibitor of caspase, suppresses apoptosis of neuritin-silenced RSCs, indicating that neuritin may be located upstream of caspase. 4.LY294002, a inhibitor of IGF-1/PI3 K, inhibits the effect of IGF-1 on prevention of apoptosis of RSCs but not the neuriti-over-expressed RSC under high glucose, indicating that neuritin may be located downstream of IGF-1/PI3 K and may mediate some of the effects of IGF-1. |
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