| Objective:Diabetic encephalopathy is a neurological complication of diabetes,and its pathogenesis is unclear.Studies believe that Alzheimer’s disease(AD)and DE have similar pathogenesis,therefore,AD is considered to be brain-specific T3 D.Current studies believe that insulin receptors and downstream signaling pathways play a key role in the occurrence and development of DE.Current studies believe that the insulin receptor and downstream signaling pathways play a key role in the occurrence and development of DE,and CLC-3,a member of the CLC family of anion channels and transporters,is closely related to the secretion and processing of insulin.Here,we investigate the changes and putative roles of CLC-3 in diabetic encephalopathy.Methods:To this aim,first we simulated the high glucose environment in HT-22 cells and confirmed the high expression of CLC-3 and other related molecules in the hippocampus of the DE rat model;secondly,we combined lentivirus transfection and adeno-associated virus stereotactic surgery And other experimental methods to change the expression level of CLC-3 in HT-22 hippocampal cell line and rat model hippocampal CA1.Through a variety of experimental methods such as morphology,molecular biology and behavior,the relationship between CLC-3 and insulin receptor signaling pathway molecules is clarified,and whether CLC-3 affects diabetic brain diseases by regulating insulin receptor signaling and its downstream pathways happened.Results:1.CLC-3 is highly expressed in HT-22 cells stimulated by hyperglycemiaCompared with the normal group of HT-22 hippocampal cells,the expression of CLC-3 molecules in HT-22 hippocampus cells was significantly increased after high glucose culture(P <0.05).In addition,compared with the control group,the expressions of APP,BACE1,Tau,and p-Tau in the high glucose group were significantly increased,and the IR was significantly decreased(all P < 0.05).QRT-PCR detects the mRNA expression levels of CLC-3,BACE1,APP,Aβ and IR,and the results are consistent with protein gel electrophoresis experiments,and the PI3K/AKT/GSK3β signaling pathway is also significantly inhibited under high glucose conditions(all P <0.05).2.Inhibiting the expression of CLC-3 can improve the down-regulation of molecules such as p-PI3 K,p-AKT,p-GSK3β and Tau caused by high glucoseCompared with the high glucose group,the molecular level of CLC-3 in the HG+ShCLC-3 group was significantly down-regulated(P < 0.05).In addition,the molecular levels of p-PI3 K,p-AKT and p-GSK3β were significantly increased compared with the high glucose group(all P <0.05).And QRT-PCR found that after down-regulating the expression of CLC-3,BACE1,APP and Aβ were significantly reduced compared with the high glucose group,and IR was significantly increased(all P <0.05).The CCK-8experiment showed that the OD value and cell viability of the cells in the high glucose group decreased significantly after 72 hours compared with the normal group.After knocking down CLC-3,the OD value and cell viability of the cells were significantly improved.The cell survival rate and apoptosis rate of the HG group were significantly different from those of the control group.After knocking down CLC-3,cell survival rate and apoptosis rate were significantly improved.3.Overexpression of CLC-3 aggravates the inhibition of PI3K/AKT/GSK3βsignaling pathway caused by high glucoseCompared with the HG group,the level of CLC-3 in the Ad-CLC-3+HG group was further significantly increased.Ad-CLC-3 further reduced the protein levels of p-PI3 K,pAKT and p-GSK3β which were down-regulated due to high glucose(all P<0.05).Compared with the HG group,the expression of APP,BACE1,Tau and pTau in HT-22 cells were also significantly increased,while the expression of IR was significantly decreased(all P<0.05).The CCK-8 experiment showed that,compared with the control group,the OD value and cell viability of the Ad-CLC-3+HG group were significantly different from those of the HG group.In addition,the apoptosis experiment also found that the apoptosis rate of HT-22 cells in the Ad-CLC-3+HG group was higher than that of the HG group,and the cell survival rate was also much lower than that of the HG group.4.Cl-channel blockers can improve the apoptosis induced by high glucose in HT-22 cells and reactivate the PI3K/AKT/GSK3β signaling pathwayThe expression level of CLC-3 molecules in the NPPB group was significantly lower than that in the control group;the protein expression levels of p-PI3 K,p-AKT and pGSK3β in the HT-22 hippocampus cells of the NPPB group were significantly higher than those in the high glucose group(P < 0.05).In addition,after using NPPB,BACE1,APP,Tau,pTau and IR in HT-22 cells were significantly different from those in the high glucose group(P <0.05).Flow cytometry experiments found that NPPB can also significantly improve the apoptotic rate and survival rate affected by high glucose(P<0.05).5.CLC-3 is highly expressed in the hippocampus of rats with diabetic encephalopathyCompared with the control group,the expression of CLC-3 in the DE group was significantly increased.In addition,the expressions of APP,BACE1,Tau,and p-Tau in the DE group were up-regulated,and the IR was significantly reduced(P <0.05).In addition,the levels of p-PI3 K,p-AKT and p-GSK3β in the hippocampus of the DE group were also significantly lower than those in the control group(P <0.05).Immunofluorescence staining showed that CLC-3,IR,and Tau all showed obvious co-localization.6.Inhibition of CLC-3 can improve STZ-induced cognitive impairment in DE rats.Compared with the DE and DE+Sh-EGFP groups,the expression level of CLC-3 in the hippocampus of the DE+Sh-CLC-3 group was significantly reduced,and p-PI3 K and p-AKT in the hippocampus of the DE+Sh-CLC-3 group And the expression level of pGSK3β molecule was also significantly different compared with DE group rats(P <0.05).In addition,the expressions of Tau,pTau,APP and BACE1 in the hippocampus of the DE+Sh-CLC-3 group were also significantly down-regulated,while the IR increased significantly(P <0.05).The water maze test showed that on the fourth day of training,the escape latency of the DE group was higher than that of the control group,and the escape latency of the DE+Sh-CLC-3 group was significantly different than that of the DE group(P <0.05).In the space exploration experiment,the number of times the DE+Sh-CLC-3group crossed the platform in the target quadrant,the time in the target quadrant and the time in the opposite quadrant were significantly different from those in the DE group(P<0.05),and there was no significant difference in the swimming speed of each group during the experiment.Conclusions:Our research suggests that CLC-3 can be used as a key molecule,which may affect the spatial cognition of diabetic encephalopathy by regulating insulin receptor signaling and downstream PI3K/AKT/GSK3β signaling pathways. |
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