| Alzheimer’s disease(AD)is the main type of dementia.Due to its complex etiology and pathogenesis,the research and development of drugs for the reversal and radical cure of AD have not been satisfactory,and the initial event of AD’s onset deserves more attention.Disorders of brain glucose metabolism and transport are initial events that affect cognitive function.More evidence indicates that AD is not only a neurodegenerative disease,but also a metabolic disease.AD belongs to the category of"dementia" in Traditional Chinese medicine,and its pathogenesis is"root vacuity and tip repletion".This deficiency mainly refers to the deficiency of the five zang organs,and the standard solid refers to phlegm turbidity and blood stasis."Heart governing blood and vessels","Heart matching fire",heart powers the body.The spleen-stomach is the source of acquired constitution,and the source of qi and blood.As the main source of energy required by the human body,glucose is associated with energy metabolism in a sense "from the heart and spleen" and dementia.As a characteristic pathological change of AD,Aβ plaque is a tangible evil,which is mainly responsible for phlegm turbidity and blood stasis.Whether the neuroprotective mechanism of Shenzhiling oral liquid(SZL)can improve cerebral glucose metabolism disorder is worth exploring.Objective In this study,based on brain glucose metabolism,the effects of SZL on brain glucose uptake,transport and glycolysis,as well as insulin signal transduction pathway InR/PI3K/Akt/GSK3β in APP/PS1 mice were investigated through a whole animal experiment.In vitro cell experiments were conducted to investigate the PI3K/Akt/GSK3β pathway in Aβ42-injured SH-SY5Y cells and the regulation of glucose transporter after blocking the pathway to further verify the molecular mechanism of the improvement of glucose metabolism in the brain by SH-SY5Y cells.Methods1.In vivo experiment:(1)Grouping administration:45 male 3-month-old APP/PS1 mice were randomly divided into model group(0.5%CMC),donepezil group(0.92 mg/kg/d)and SZL group(2.9 mL/kg/d),with 15 mice in each group,control group consisted of 15 C57BL/6J mice of the same age(same as model group),which were given intragastric gavage for 3 months.(2)Behavioral evaluation:Y maze,Morris water maze,and diving platform were used to evaluate the cognitive effects of SZL on APP/PS1 mice.(3)Characteristic pathological change:HE,immunohistochemical staining and WB were used to evaluate the effect of SZL on the characteristic pathological change of Aβ in APP/PS1 mice.(4)Ultrastructure observation:The ultrastructural changes of microvessels,astrocytes and neurons in the hippocampus of mice were observed by transmission electron microscopy.(5)Micro-PET detection:Micro-PET showed that the glucose uptake in the hippocampus and cortex(parietal lobe,temporal lobe,frontal lobe,entorhinal cortex,posterior cingulate cortex)was not statistically significant,but the results indicated that the glucose uptake in the brain region of the model group was decreased,while the glucose uptake in the SZL group was improved to varying degrees.(6)Immunohistochemistry and WB test:Immunohistochemistry,WB were used to observe the effects of SZL on insulin signal transduction PI3K/Akt/GSK3β pathway,glucose transporter GLUT1 and GLUT3 expression in APP/PS1 mice.(7)RT-qPCR detection:RT-qPCR was used to observe the effects of SZL on PI3K/Akt/GSK3βpathway,glucose transporter GLUT1,and key enzymes of hippocampus glycolysis in APP/PS1 mice.2.In vitro experiment:(1)Non-target metabolomics of Chinese medicine:UHPLC-QE-MS was used to identify the active components of SZL and its drug-containing serum.(2)Establishment of Aβ42 damaged cell model:Establishment of AD model in SH-SY5Y cells damaged by Aβ42 in vitro.(3)Cell aging and dose response curve measurement:CCK8 was used to screen the safe and effective dose of drug-containing serum of SZL on SH-SY5Y cells.(4)WB,RT-qRCR and immunofluorescence detection:WB,RT-qRCR and immunofluorescence were used to study the effects of drug containing serum of SZL on insulin signal transduction PI3K/Akt/GSK3β pathway and glucose transporter in Aβ42-injured SH-SY5Y cells.(5)WB detection:The regulatory role of insulin signal transduction pathway PI3K/Akt/GSK3β in glucose metabolism and transport was confirmed by PI3K/Akt/GSK3βinhibitor LY294002 and GSK3β inhibitor LY2090314.Results1.In vivo experiment:(1)Behavioral test results:Y maze results showed that the spontaneous alternation rate of model group was lower than that of normal group(P<0.01).The spontaneous alternation rate of mice in donepezil group and SZL group was increased(P<0.05).Morris water maze results showed that the average swimming distance and average escape latency of model group were increased compared with the control group on the 3rd to 5th day(P<0.01).On day 4 of the test,the mean swimming distance and mean escape latency of mice in donepezil and SZL groups were decreased compared with those in model group(P<0.05).After removal from the platform,the residence time across the platform and target quadrant of the model group was decreased compared with the control group(P<0.01).The residence time in the target quadrant of mice in donepezil and SZL groups was increased compared with that in model group(P<0.05 or P<0.01).The results showed that the number of specific errors in model group was higher than that in control group,and the incubation period was shortened(P<0.05).Compared with model group,donepezil group and SZL group had fewer errors and increased latency(P<0.05).(2)Characteristic pathological changes:Immunohistochemistry showed that Aβ plaques in hippocampus and cortex of model group were increased compared with normal group(P<0.05,P<0.01).Compared with model group,the expression of Aβ plaque and Aβ42 protein in hippocampus and cortex of donepezil group and SZL group decreased(P<0.05,P<0.01).WB results showed that compared with the normal group,the expression of Aβ42 protein in the hippocampus and cortex of model group was significantly increased(P<0.01),and the expression of Aβ42 protein in the hippocampus and cortex of model group was significantly decreased in the donepezil and SZL groups(P<0.01).(3)Ultrastructural results:Transmission electron microscopy results showed that the model group mice hippocampal CA1 region neuron pyrexia,neuronal mitochondria broken,cristae fracture,microvascular and astrocytes obvious edema,deformation;Compared with model group,SZL and donepezil groups had different degree of improvement.(4)Micro-PET results:The glucose uptake in the hippocampus and cortex(parietal lobe,temporal lobe,frontal lobe,entorhinal cortex and posterior cingulate cortex)of each group showed a decreasing trend in the model group,while the glucose uptake in the brain region of the SZL group was improved in different degrees(P>0.05).(5)Immunohistochemical results:Immunohistochemical results showed that the number of InR,IRS2,PI3K,Akt and p-GSK3β positive cells in the hippocampus of model group was significantly lower than that of control group(P<0.05 or P<0.01).Compared with model group,the number of positive cells of above indexes in donepezil and SZL groups was significantly increased(P<0.05 or P<0.01).The number of GSK3β positive cells in model group was significantly increased(P<0.01),and the number of GSK3β positive cells in donepezil and SZL groups was significantly lower than that in model group(P<0.01).Compared with the control group,the number of GLUT3 positive cells in the hippocampus of model group was significantly decreased(P<0.01).Compared with model group,the number of GLUT3 positive cells in the hippocampus of donepezil and SZL groups was significantly increased(P<0.05 or P<0.01).(6)WB results:Compared with the control group,hippocampal PI3K,Akt,p-Akt and p-GSK3β in model group were all decreased,and IRS2,PI3K and p-Akt were significantly decreased(P<0.05 or P<0.01).Compared with model group and donepezil group,the above proteins were increased,and p-Akt and p-GSK3β were significantly increased(P<0.05 or P<0.01).However,the expression of GSK3β protein in hippocampus in model group was significantly higher than that in control group(P<0.01),and the expression of GSK3βprotein in donepezil and SZL groups was significantly lower than that in model group(P<0.01).Compared with the control group,the expression of GLUT1 and GLUT3 protein in the hippocampus of model group was decreased,and there were significant differences in the levels of GLUT1 and GLUT3(P<0.01 or P<0.05).Compared with model group,the expression of GLUT1 and GLUT3 protein in hippocampus of donepezil group and SZL group increased,and GLUT1 increased significantly(P<0.01),while GLUT3 in SZL group increased significantly(P<0.05).(7)RT-qPCR results:Compared with the control group,the expression of InR,IRS2,GSK3βand GLUT1 genes in model group were significantly decreased(P<0.01).Compared with model group,InR mRNA,IRS2 mRNA,GSK3β mRNA and GLUT1 mRNA in donepezil and SZL groups were significantly increased(P<0,05 or P<0.01).Compared with the control group,the expression levels of key enzymes of glucose metabolism in hippocampus of the model group were decreased,including HK1 mRNA,CoXIV mRNA,ATPase mRNA and AMPK mRNA,and the CoⅩⅣ mRNA and AMPK mRNA were significantly decreased(P<0.05 or P<0.01).Compared with model group,the expressions of HK1 mRNA,CoⅩⅣmRNA,ATPase mRNA and AMPK mRNA in SZL group were up-regulated to different degrees(P<0.05 or P<0.01).2.In vitro experiment:(1)UHPLC-QE-MS result:In this study,46 kinds of blood components were identified by UHPLC-QE-MS.(2)Results of cell aging and dose response curve:CCK8,flow cytometry and cell morphology experiments were used to determine the incubation time of 5 μm aged Aβ42 cells for 48h to establish an in vitro model of AD.CCK8 method was used to screen the optimal intervention time and concentration of 15%drug-containing serum for 48h on Aβ42 injured SH-SY5Y cells.(3)WB results:The expression of insulin signaling pathway PI3K/Akt/GSK3β signaling pathway related proteins and glucose transporter proteins in Aβ42-injured SH-SY5Y cells decreased.The protein expressions of PI3K,Akt,p-Akt,GLUT1 and GLUT3 were significantly decreased(P<0.05 or P<0.01).The drug-containing serum of SZL could improve the expression of insulin signaling pathway PI3K/Akt/GSK3β signaling pathway related proteins and glucose transporter proteins,and the protein expressions of Akt,p-Akt,GLUT1 and GLUT3 were significantly increased(P<0.05 or P<0.01).(4)RT-qPCR results:The Aβ42-injured SH-SY5Y cells could down-regulate the expression of PI3K mRNA,Akt mRNA,GSK3β mRNA,GLUT1 mRNA and GLUT3 mRNA.Akt mRNA,GSK3β mRNA,GLUT1 mRNA and GLUT3 mRNA were significantly down-regulated(P<0.05 or P<0.01).The expression of the above genes could be up-regulated in the drug-containing serum of SZL,among which Akt mRNA,GSK3β mRNA,GLUT1 mRNA and GLUT3 mRNA were up-regulated significantly(P<0.05 or P<0.01).The use of PI3K/Akt inhibitor LY294002,GSK3β inhibitor LY2090314 and the combination of the two inhibitors could reverse the up-regulation of GLUT1 and GLUT3 protein expression in drug containing serum of SZL to different degrees.ConclusionImproving glucose uptake,transport and glycolysis in the brain may be the pathway of the neuroprotective effects of SZL,and its potential molecular mechanism may be related to the improvement of insulin signal transduction pathway disorder in early AD. |
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