| Background Diabetic encephalopathy(DE)is a central neuropathy caused by diabetes mellitus(DM),which mainly manifests as cognitive dysfunction such as learning and memory.For a long time,its mechanism has not been elucidated,and there is a lack of specific and effective treatment methods.Therefore,to clarify its pathogenesis and find effective drug treatment targets is one of the key clinical issues that need to be addressed in DE research.Recently,it has been found that cognitive dysfunction in DE is closely related to hippocampal synaptic plasticity,mainly by affecting the neurotransmitter release in presynaptic membrane,glutamate receptor function and distribution in postsynaptic membrane,and inducing imbalance of intersynaptic calcium homeostasis,which ultimately leads to decreased long-term potentiation(LTP)and increased long-term depression(LTD),leading to cognitive impairment.However,the underlying mechanisms that induce these changes in synaptic plasticity are unclear.Actin,as one of the cytoskeletal components,is the basis for maintaining synaptic morphology.Cofilin is an actin-binding protein that rapidly regulates the polymerization or depolymerization of actin fibers,and LIM-kinase 1(LIMK1)protein plays an important role in regulating neuronal morphology and synaptic growth by regulating cytoskeleton formation through a phosphorylation pathway.Furthermore,the Limk/Cofilin pathway is involved in synapticα-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid(AMPA)receptor transport and accumulation by influencing synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid(AMPA)receptor after LTP induction.regulation of synaptic plasticity.Lipin1 is a member of the Lipin family of proteins,a gene closely related to glucose and lipid metabolism,and alterations in Lipin1 expression and gene variants have been associated with insulin resistance and the development of type 2 diabetes.Its physiological function depends mainly on the activity of phosphatidic acid phosphatase(PAP),which can produce diacylglycerol(DAG)from phosphatidic acid(PA),and DAG can make protein kinases D(PKD).DAG can phosphorylate protein kinases D(PKD),which can be involved in actin reorganization and actin cytoskeleton-driven cell-directed migration.In addition,PKD has an important role in early,middle and late neuronal development.We have shown peripheral neuropathy and cognitive dysfunction in fatty liver dystrophy(fld)mice with Lipin1 deficiency,and we also found decreased Lipin1 expression in diabetic peripheral neuropathy(DPN)and hippocampal CA1 in DE rats.So what is the role of Lipin1 in DE rats?We explored whether Lipin1 is involved in the pathogenesis of DE through the PKD/Limk/Cofilin signaling pathway regulating synaptic plasticity by molecular biology techniques.Part Ⅰ Effect of Lipin1 on the activity of PC12 cells under different glucose concentrationsObjective Observe the activity of Pc12 cells under different glucose concentrations,the proliferative activity of PC 12 cells after up-and down-regulation of Lipin1 expression by infection with lentivirus was observed.Methods 1.CCK-8 detects the activity of pc 12 cells under different glucose concentration culture conditions.The pc12 cells were incubated at different glucose concentrations(25,50,75,100,125,150 mmol/L)for 24h and 48h(the same concentration of mannitol was used as control),and CCK-8 assayed the activity of pc12 cells.25 mmol/L was chosen as the normal glucose concentration.2.Electron microscopy and hoechst 33342 staining were used to observe the morphological changes of PC 12 cells in control and high glucose groups.3.Quantitative Real-Time Polymerase Chain Reaction(QPCR)and Western Blot(WB)were used to detect the difference of lipinl expression in pc 12 cells under normal and high glucose culture conditions4.Transfection of pc 12 cells with lipinl lentivirus and verification of transfection efficiency.The lipinl lentivirus(Lv-Lipin1ShRNA、Lv-Lipin1ShRNA-Con、Lv-Lipin1、Lv-Lipin1-Con)were transfected into PC 12 cells.The transfection efficiency was further verified by qPCR and western blot for Lpin1 mRNA and protein content.5.The pc 12 cells transfected with lentivirus were grouped into HG+Lv-Lipin1(high glucose+Lv-Lipin1),HG+Lv-Con(high glucose+Lv-Con),NG+Lv-Lipin1 ShRNA(normal glucose+Lv-Lipin1ShRNA),NG+Lv-Con(normal glucose+Lv-Con).CCK-8 was used to detect the activity of each group of cells.Results1.With the increase of glucose concentration and time,the activity of PC 12 cells showed a decreasing trend.Compared with the control group,the difference in cell activity was greater at 100 mM/48 h(P<0.01)than at 75 mM/48 h(P<0.05),so 25 mmol/48 h was selected as the normal control group condition(normal glucose,NG)and 100 mM/48 h as the high glucose group condition(high glucose,HG)for subsequent experiments.2.The apoptosis of cells in HG group was increased compared with NG group.3.The expression of Lipin1 mRNA and protein was significantly reduced in HG group cells compared with NG group(P<0.01).4.CCK-8 detects the activity changes of pc12 cells under normal and high glucose after transfection of lipinl lentivirus.The activity of pc12 cells in the HG+Lv-Lipin1 group was increased compared with the HG+Lv-Con group(P<0.01).The activity of pc 12 cells in the NG+Lv-Lipin1 ShRNA group was decreased compared with the NG+Lv-Con group(P<0.01).Conclusions 1.The activity of PC 12 cells was negatively correlated with sugar concentration and time,and the reduction of PC 12 cell activity was accompanied by the reduction of Lipin1 expression.2.The overexpression of Lipin1 has a protective effect on the decrease of PC 12 cell activity induced by high glucose,while reducing the expression of Lipin1 in PC12 cells will result in the decrease of PC 12 cell activity under normal glucose conditions.Part Ⅱ Study on the mechanism of Lipin1 in cognitive dysfunction of rats with diabetic encephalopathyObjective To investigate whether Lipin1 regulates synaptic plasticity through PKD/Limk/Cofilin signaling pathway involved in the pathogenesis of diabetic encephalopathy.Methods 1.A type 1 diabetic rat model was constructed.Water maze experiments were performed to screen the successfully modeled DE rats.2.Detect the difference of Lipin1 expression in the CA1 region of wild-type rats and DE rats.3.Lipin1 lentivirus was stereotaxically injected into CA1 region of rat hippocampus.The groups were WT+Lv-Lipin1ShRNA,WT+Lv-Con,DE+Lv-Lipin1,DE+Lv-Con,and 2 weeks later,the behavioral experiments were performed to detect the changes of cognitive function in each group of rats.4.Electron microscopy and Golgi staining were performed to observe the changes in the number of synapses and the morphology and density of dendritic spines in the CA1 area of each group of rats.5.To detect the DAG level,p-Ssh1,p-PKD,p-Limk1,p-Cofilin and Syn protein expression in CA1 area of rats in each group.Results 1.From the 3rd day of STZ injection,rats in the DM group showed increased blood glucose(P<0.01)and decreased body weight(P<0.01)compared with the WT group.2.Morris water maze experiment suggested that after 12 weeks of STZ injection,the cognitive function of rats in DM group decreased compared with that in WT group.3.qPCR and western blot detected the expression of lipinl in CA1 region of wild-type rats and DE rats.The content of Lipin1 mRNA and protein was significantly reduced in diabetic rats compared with wild-type rats(P<0.01).4.In the open field test and new object experiments,the activity time and discrimination index(DI)in the central region were reduced in the DE+LV-Con(P<0.01)and WT+LV-Lipin1 ShRNA groups(P<0.05)compared with the WT+LV-Con group.The activity time and DI in the central region were increased in the DE+LV-Lipin1 group(P<0.05)compared with the DE+LV-Con group.In the Morris water maze experiment,the escape latency of the DE+LV-Con group increased compared with that of the WT+LV-Con group on days 3(P<0.01)and 4(P<0.05)of the localization navigation period;the escape latency of the DE+LV-Lipin1 group decreased compared with that of the DE+LV-Con group.The difference between the WT+LV-Lipin1 ShRNA group and WT+LV-Con group was not statistically significant.During the spatial exploration period,the DE+LV-Con and WT+LV-Lipin1 ShRNA groups traversed the platform less frequently than the WT+LV-Con group(P<0.01),and the DE+LV-Lipin1 group traversed the platform more frequently than the DE+LV-Con group(P<0.05).5.The number of synapses and dendritic spine density in WT+LV-Lipin1 ShRNA and DE+LV-Con groups were lower than those in WT+LV-Con group(P<0.01).And the number of synapses and dendritic spine density were increased in the DE+LV-Lipin1 group compared with the DE+LV-Con group(P<0.01).6.DAG levels,P-PKD,P-Limk1,P-Cofilin,p-Sshl and Syn protein expression levels in the CA1 region of the rat hippocampus were decreased in the WT+LV-Lipin1 ShRNA group and the DE+LV-Con group;whereas the levels of these proteins were increased in the DE+LV-Lipin1 group compared with the DE+LV-Con group.The differences were all statistically significant.Conclusions 1.Compared with wild-type rats,the expression of Lipin1mRNA and protein in the hippocampal CA1 area of diabetic rats was decreased.2.Silencing Lipin1 expression in the CA1 region of normal rats resulted in cognitive dysfunction,as seen by a decrease in the number of neuronal synapses and dendritic spines,while overexpression of Lipin1 in the CA1 region of DE rats resulted in improved cognitive function and a decrease in the number of neuronal synapses and dendritic spines.3.Lipin1 expression in the hippocampus of rats with diabetic encephalopathy may be involved in the development of diabetic encephalopathy by regulating synaptic plasticity in the hippocampus through the PKD/Limk/Cofilin signaling pathway. |
