| BackgroundDiabetic encephalopathy is an increasingly recognized complication of diabetes mellitus. The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway was shown to be critical for the neuropathogenetic process of cognitive dysfunction in diabetes. Growth factor receptor-bound protein 10 (Grb10) is an intracellular adapter protein that binds directly to IGF-1R and negatively regulates the IGF-1R signaling pathway. Grb10 interacting GYF protein 1 (GIGYF1) is a novel protein that links to the N terminus of Grb10. When over-expressed, GIGYF1 can augment the IGF-1R signaling pathway. However, the in vivo molecular mechanism of endogenous Grb10 in regulating the IGF-1R signaling pathway and its downstream signaling pathway in the brain is not clear.ObjectivesThe objective of this study was to observe the effect of Grb10-shRNA on behavioral changes and pathological changes, to further dissect the underlying molecular mechanisms of IGF-1R signaling pathway and its downstream signaling pathway in the brain that is involved in the development of diabetic encephalopathy.MethodsDiabetes mellitus (DM) was induced by intraperitoneal injection of streptozotocin (60 mg/kg body weight) dissolved in 0.1M sodium citrate-hydrochloric acid buffer solution (pH 4.5). Three days after STZ injection, fasting blood glucose levels were obtained from the tail vein of rats. Rats with a fast blood glucose level of 18 mmol/L or above were declared diabetes and selected to the DM group. An equal number of normal rats were used for comparison. Rats were randomly assigned to six groups (n=10 animals per group), i.e. control group (con), control group treated with sham-knocked down (con+0), control group treated with Grb10-knocked down (con+shRNA), DM, DM group treated with sham-knocked down (DM+0), DM group treated with Grb10-knocked down (DM+shRNA). The fasting blood glucose concentrations were measured once a week.3 months after the surgery, cognitive function of all rats was observed in morris water maze (MWM) test. Then, we evaluated the changes of ultrastructure, pathology and histology in the hippocampus. And, the expression of Grb10, GIGYF1, IGF-1R, IRS1, IRS2, Akt and Erk1/2 levels were detected by qRT-PCR and western blot.Results1. Based on the MWM data, there was no significant difference in the mean escape latency among all the groups before and just after the surgery, as well as the result of the time in target quadrant and platform crossing (P>0.05). Physical exercise produced a significant decrease in time to reach platform in diabetic rats with Grb10 knockdown compared with diabetic rats 3 months after surgery (P<0.01). According to the probe trials, the frequency of platform crossing and the time spent in the target quadrant demonstrated significant increase in the control and DM+shRNA group compared with the DM group 3 months after the surgery (P<0.01). Moreover, there was no difference between the DM+shRNA group and the control group (P>0.05).2. The state of chronic hyperglycemia could lead to changes in cell morphology and tissue ultrastructure. The results of the electron microscopy showed that rats with diabetic encephalopathy demonstrated a significant loss in the number of spine synapses in the hippocampal CA1 areas compared to the control group. However, the quantity and morphology of neurons in diabetic rats with Grb10 knockdown remained was similar to normal hippocampal structure of rats.3. Related pathological and histological changes, including a decrease neuron numbers and an increase in neuronal apoptosis, were observed in the hippocampus of the DM group. The morphology of the tissues from the diabetic group showed obvious changes compared with the control group, but appeared as normal in the DM+shRNA group. Immunohistochemical studies indicated that the expression level of Grb10 in the DM group was significantly higher than that of the control and the DM+shRNA group (P<0.01), and there was no marked difference between the control and the DM+shRNA group (P>0.05).4. We assessed the expression level of Grb10 mRNA in the hippocampus following the application of Grb10-shRNA. Grb10 mRNA level significantly increased in the DM group in comparison with the control group (P<0.01), and was restored to the control levels by the Grb10 knockdown in the DM group. Similarly increased level of GIGYF1 mRNA was observed in DM group, which was higher than in the control and DM+ shRNA group (P<0.01). IGF-1R mRNA level significantly decreased in the DM group compared with the control and DM+shRNA group (P<0.05). Additionally, there were no statistical differences in the mRNA levels between the control group and the DM+shRNA group (P>0.05).5. We then examined the effect of Grb10 knockdown on the protein expression of IGF-1R signaling pathway and its downstream signaling pathway in the hippocampus. Grb10 and GIGYF1 protein expression levels significantly increased in diabetic rats compared with the non-diabetic control (P<0.01). Application of Grb10 shRNA to the hippocampus in vivo using stereotactic technology could efficiently down-regulate Grb10 and GIGYF1 protein expression levels in diabetic rats. In response to IGF-1, the phosphorylation of IRS 1 and IRS2 protein levels were decreased in the DM group, but showed a significant increase in IRS phosphorylation in Grb10-deficient diabetic rats (P<0.01 or P<0.05). In addition, the phosphorylated IGF-1R protein level of diabetic group was lower in comparison with the control, and a significant increase in IGF-1R phosphorylation was observed in diabetes rats after Grb0-shRNA treatment (P<0.05). Meanwhile, Grb10 knockdown resulted in enhanced IGF-1R-mediated Akt/PKB phosphorylation relative to the DM group ((P<0.01). Our data also demonstrated that IGF-1 stimulated phosphorylation of Erkl and Erk2 were increased as a consequence of Grb10 knockdown without changes in total Erkl/2 protein levels(P<0.01 or P<0.05). Additionally, there were no statistical differences in the protein levels between the control group and the DM+ shRNA group (P>0.05).ConclusionsRats under chronic high blood glucose pressure would undergo obvious behavioral changes and induce Grb10 over-expression in the hippocampus. The level of Grb10 expression could be efficiently down-regulated by the application of lentiviral vector-delivered Grb10 shRNA to the hippocampus. In this study, we demonstrate that interference with endogenous Grb10 expression could decrease GIGYF1 activation, and increase IGF-1-stimulated IGF-1R, IRS, Akt, and Erkl/2 activation as well as cognitive function in rats with diabetic encephalopathy. We also speculate that Grb10 and GIGYF1 may act cooperatively to regulate IGF-1 and its receptor-induced signaling pathways as well as related pathological changes in the brain. Additionally, down-regulation of Grb10 expression could decrease hippocampal neuronal apoptosis caused by long-term hyperglycemia, suggesting that over-expression of Grb10 is correlated with neuron apoptosis in the hippocampus. We conclude that lentivirus shRNA Grb10 targeting the hippocampus can reduce cognitive impairment induced by chronic hyperglycaemia and enhance the IGF-1R signaling pathway and its downstream signaling pathway in rats with diabetic encephalopathy. Thus, we infer that Grb10 could be inferred as a key molecule in the regulation of neural functions in diabetic encephalopathy. Targeted manipulation of Grb10 expression may provide a novel approach for the effective treatment of diabetes-associated cognitive disorders. |
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