| Background:Diabetic encephalopathy(DE)is a complication of the central nervous system of diabetes,the main clinical manifestation is cognitive dysfunction,and abnormal phosphorylation of tau protein in the brain is considered to be one of its main pathological features,but the specific pathogenesis is not completely clear yet.Due to the continuous advancement of medical technology,the life span of diabetic patients has been significantly extended,which has also led to a continuous increase in the prevalence of DE.Therefore,it is very important for us to discover effective drugs for the treatment of DE and its possible pathogenesis.Classical medical records that Coptidis Rhizoma has the effect of treating "diabetes" and "dementia".Modern research has also confirmed that Coptidis Rhizoma has significant effects in the treatment of diabetic complications and neurodegenerative diseases.However,the research mainly focuses on berberine,about other alkaloids from Coptidis Rhizoma in this area has not been reported.Purpose:In this study,the primary neurons induced by high glucose were used as the DE cell model.We want to systematically systematically isolate and screen the alkaloid monomers that are effective for DE from Coptidis Rhizoma,and then evaluate the efficacy in the db/db mice.Finally,the related mechanism was preliminarily discussed in vivo and in vitro.Methods:1.Extraction and separation of alkaloids from Coptidis Rhizoma and evaluate their activity to improve DEThe total alkaloids were extracted from Coptidis Rhizoma by acid-water percolation method,and then extracted with petroleum ether,dichloromethane and n-butanol respectively.The content of alkaloids in each part was analyzed by HPLC,and the main alkaloid parts were subjected to silica gel column chromatography.Separate,divide the total alkaloids into several different sections according to the polarity of the alkaloids.Using high concentration of glucose to induce primary neurons to establish a DE cell model,using phosphorylated tau protein as an indicator of drug efficacy,and then using the obtained main alkaloid segments to intervene in neurons to screen out the most active alkaloid segments.Furthermore,gel column chromatography was used to separate and purify the effective passages to obtain the main alkaloid monomers.The structure of the alkaloids was determined by a nuclear magnetic resonance instrument,and the purity of the alkaloids was measured by HPLC.Finally,evaluate the efficacy of drugs in the established DE cell model.2.The improvement effect of epiberberine on cognitive impairment in db/db micedb/db mice were selected as the DE model,and db/m mice were used as controls.The mice were randomly divided into 5 groups,each with 8 animals: control group db/m,model group db/db,berberine group(200 mg/kg),epiberberine low-dose group(100 mg/kg),epiberberine high-dose group(200 mg/kg),continuous administration for12 weeks.During the administration period,the weekly body weight and fasting blood glucose of the mice every two weeks were recorded.After the administration,the spatial learning and memory ability of the mice was tested by water maze experiment and new object recognition experiment,and the pathological damage of the mice hippocampus and the changes in the number of neurons were evaluated by HE staining and Nissl staining,and immunohistochemistry staining and western blotting were used to detect the expression and localization of tau protein in the hippocampus of mice.3.The study of epiberberine on IGF1R/PI3K/AKT/GSK3β pathwayAccording to literature reports,the possible target of epiberberine to improve DE was found,and the molecular docking software Le Dock was used to analyze the binding of epiberberine and IGF1 R.Finally,using RT-qPCR,immunofluorescence and western-blot methods to study targets and signal pathways in animals and cell models.And further add inhibitors to neurons to study the molecular mechanism of epiberberine to improve DE.Results:1.Epiberberine significantly reduces the hyperphosphorylation of tau protein in neurons induced by high glucoseCoptidis Rhizoma was extracted with acid water and neutralized with calcium oxide,and then extracted with petroleum ether,dichloromethane and n-butanol.It was found that its alkaloids were mainly concentrated in the n-butanol part.The n-butanol part was separated by column chromatography to obtain A,B,C,D,E and F main alkaloid segments.After the primary neurons were cultured in 45 mM glucose for 24 h,the tau protein was phosphorylated significantly at Ser396 and Ser404.Then,it was found that the C-segment alkaloids can significantly reduce the level of tau protein phosphorylation in neurons induced by high glucose.After further separation and purification of the C-segment alkaloids,a major alkaloid monomer was obtained,which was identified as epiberberine by its structure.The purity is 98 % measured by HPLC.Through cell experiments,it was found that epiberberine can significantly reduce the phosphorylation level of tau protein in neurons induced by high glucose in a dose-dependent manner.2.Epiberberine significantly cognitive impairment and pathological damage in db/db miceAfter 12 weeks of administration,the body weight and fasting blood glucose of db/db mice decreased significantly.Behavioral results showed that compared with db/m mice,the learning and memory ability of db/db mice was significantly reduced,while epiberberine significantly improved the cognitive function of db/db mice.The results of HE staining showed that the hippocampal DG area of db/db mice showed obvious damage,deep cracks and enlarged intercellular spaces,while there was no obvious abnormality in the tissue structure of the hippocampal DG area of mice in the epiberberine treatment group,and the neuronal cells were arranged regularly,and no obvious cracks.The results of Nissl staining showed that the neurons in the hippocampal DG area of db/db mice were significantly damaged,the cell membrane shrank and ruptured,the number of Nissl bodies was small and fuzzy,and the number of neurons was significantly reduced,while the epiberberine treatment group the morphology of neurons in the DG area of the mice hippocampus is relatively complete,with clear outlines,and a relatively large number of neurons.Western blot and immunohistochemical results showed that epiberberine significantly reduced tau protein hyperphosphorylation in the hippocampal DG region of db/db mice.In vivo experiments proved that epiberberine has a significant improvement effect on cognitive impairment and pathological damage in db/db mice,and the effect is better than berberine.3.Epiberberine reduces tau protein hyperphosphorylation through the IGF1R/PI3K/GSK3β pathwayIGF1R is the upstream target of PI3K/AKT signaling pathway,which affects PI3K/AKT signaling and GSK3β activity.Epiberberine and IGF1 R molecular docking results show that epiberberine forms intramolecular hydrogen bonds with IGF1 R protein MET-1082 and GLU-1050,and interacts with multiple amino acid molecules in the functional region of IGF1 R protein.RT-qPCR results showed that epiberberine can increase the expression of IGF1 R and IGF1 mRNA in the hippocampus of db/db mice.In addition,epiberberine also up-regulated the expression of IGF1 R,IGF1,PI3 K,p-AKT and p-GSK3β in the hippocampus and high glucose-induced neurons of db/db mice.After treatment of neurons with IGF1 R inhibitors,the inhibitors can partially block epiberberine-activated IGF1R/PI3K/GSK3β signaling and epiberberine intervention-induced tau protein phosphorylation down-regulation.Conclusion:Epiberberine has a significant improvement effect on DE cognitive impairment and pathological damage,and the effect is better than berberine.Its mechanism of action may be achieved by regulating the IGF1R/PI3K/GSK3β pathway to inhibit the hyperphosphorylation of tau protein. |
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