| Objective:Diabetic encephalopathy is one of major complications of diabetes mellitus(DM).The nerve cells injury caused by hyperglycemia is a key factor of diabetic encephalopathy in DM patients.Nerve growth factor(NGF)is the earliest discovered neurotrophic factor,which plays an important role in the growth,development,differentiation,survival of nerve cells.Recombinant human nerve growth factor(rhNGF)is highly homologous with human nerve growth factor(hNGF).In this study,PC 12 cells were used to investigate the protection effects of rh NGF in hyperglycemia induced nerve injury and its possible mechanisms.Methods:(1)To evaluate the protective effect of rhNGF:PC 12 cells or primary isolated neurons were treated with 50-150 mM glucose for 48 h or 100 mM glucose for 24-72 h,CCK-8 was used to detect the cell viability to develop high glucose induced cells injury model.CCK-8 was used to evaluate the protective effect of rhNGF based on cell injury model.(2)To evaluate the effect of rhNGF on high glucose induced apoptosis:①Hoechst 33258 staining,Tunel staining and flow cytometry assay were used to detect apoptosis.②RT-PCR was used to determine the mRNA levels of apoptosis-related factors(Puma,Bcl-2 and Bax).③Western blot was used to measure the expression of apoptosis-related proteins(cleaved caspase-3 and p53).(3)To evaluate the effects of rhNGF on high glucose induced oxidative stress:①CellROXTM Deep Red dye Reagent was used to assess the level of reactive oxygen species(ROS);ELISA kit was used to evaluate the level of 3-nitrotyrosine(3-NT).(4)To explore the possible signal pathways:(1)PC12 cells were treated with 25 ng/mL rhNGF for 0-20 min or different doses rhNGF for 5 min,and the expression levels ofrelated proteins(p-AKT、T-AKT、p-ERK 1/2、T-ER[K1/2)were detected by Westerm blot.(2)PC 12 cells were treated with/without rhNGF and high glucose,and the expression levels of related proteins(p-AKT、T-AKT、P-ERK1/2、T-ERK1/2、p-FoxO3a、T-FoxO3a)were detected by Western blot.③ PC 12 cells were pre-treated with GW441756(10μM),MK-2206(200 nM)and SCH772984(200 nM)for 1h,then co-treatment with high glucose in the absence or presence of rhNGF,CCK-8 was used to measure the cell viability.④ PC12 cells were pre-treated with MK-2206(200 nM)and SCH772984(200 nM)for 1 h before treatment with rhNGF,and the expression levels ofrelated proteins(p-AKT、T-AKT、p-ERK 1/2、T-ERK1/2)were detected by Western blot.⑤ PC 12 cells were pre-treated with MK-2206(200 nM)1h,then co－treatment with high glucose in the absence or presence of rhNGF,CellROX T,Deep Red Reagent was used to assess the level of ROS.Results:(1)PC12 cells or primary cultured neurons were treated with 100 mM high glucose for 48 h developed a stable cell injury model,and rhNGF significantly increased cell viability in a dose-dependent manner under the condition of high glucose.(2)The results of Hoechst 33258 staining,Tunel staining and flow cytometry assay show that rhNGF could effectively reduce high glucose induced apoptosis;rhNGF reversed the mRNA levels of Puma and Bcl-2/Bax;rhNGF reversed the protein expression levels of cleaved caspases-3 and p53.(3)rhNGF could effectively decrease the ROS level and 3-NT level.(4)rhNGF significantly increased the levels of p-AKT/T-AKT,p-ERK1/2/T-ERKI/2 in PC12 cells;rhNGF significantly reversed the levels of p-AKT/T-AKT,p-FoxO3a/T-FoxO3a,but the level of p-ERK1/2/T-ERK1/2 did not show significantly change in PC 12 cells under high glucose condition;GW441756(10 μM)and MK-2206(200 nM)weaken the protective effect of rlNGF in PC 12 cells,while SCH772984(200 nM)had no significant effect on the protective effect of rhNGF;MK-2206(200 nM)and SCH772984(200 nM)inhibited the activation of AKT and ERK1/2,respectively;MK-2206(200 nM)weaken the role of rhNGF inhibited the production of ROS.Conclusion:rhNGF could effectively inhibit high glucose induced apoptosis and oxidative stress,which may be related to the activation of AKT/Fox03a signaling pathway. |
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