| Background:Aging was a normal phenomenon,and the functions of tissues and organs could undergo degenerative changes.The susceptibility of cerebrovascular diseases,neurodegeneration and other diseases could be increased during the progress of aging,which could effect the quality of life in the elderly.Amd the brain seems to be more susceptible in the aging organs.The cortex and hippocampus in brain had been involved in aging pathogenesis study for decades.The cortex was extremely vulnerable to change in structures and functions that associated with cognitive decline.The hippocampus presented neurobiological alterations during the process of aging,including neuroinflammation,increased oxidative stress,altered gene expression and so on.And these alterations in hippocampus were also associated with age-related cognitive decline.Therefore,it had great significance to explore the mechanisms of aging and screen the active ingredients with neuroprotective effects from natural products for prevention of senile diseases that related to aging,and it was also important to improve the quality of life of the elderly.Scutellaria baicalensis Georgi(SBG),one of traditional herbal medicines,had the common name Huang Qin in Chinese.The roots were recognized as the medicinal parts of SBG,and compounds and extracts isolated from roots had a wide range of pharmacological activities,including antibacterial,antiviral,antioxidant,anti-inflammatory,antiaging,neuroprotective effects and other pharmacological effects.The resources of SBG in Shanxi province were extremely rich.And the stems and leaves of SBG had become a different kind tea in northern areas of China for thousands of years.Literature reported that although the content of the main components in the stems and leaves of SBG were different,there were both contain a large number of flavonoids.Feng et al found that the leaves of SBG(LSBG)and its tea had anti-aging effects based on the modle of natural aging fruit flies.However,the anti-aging mechanism of LSBG had not been elucidated.Therefore,in terms of the comprehensive utilization of resources and sustainable development,the research on the anti-aging effects and mechanisms of LSBG had important practical significance for further mining the potential pharmacological activities of SBG.Therefore,the LC-MS technology was used to analyze the changes of endogenous metabolites in hippocampus and cortical tissue samples of aging rats.Combined with literatures,glutamate metabolism as the key pathway were verified,and the content of metabolites and the activities of metabolic enzymes in this pathway were detected by using kits.In addition,the Nrf2 signaling pathway was an important regulator in the defense system that protected the brain from oxidative stress,and the expression of its downstream proteins could regulate the expression of enzymes involved in glutathione synthesis,then it affecting the process of synthesizing glutathione from glutamate.However,the mechanism of aging in the Nrf2 signaling pathway and the regulation of the expression of key proteins in this pathway by LSBG were unclear.Therefore,two aspects were detected in this study,including the glutamate metabolism pathway and Nrf2 signaling pathway.It could be provide experimental basis for further development of LSBG resources.Purpose: 1.To evaluate the anti-aging efficacy of LSBG at different dosage based on the model of D-gal-induced aging rats;2.To study the changes of endogenous metabolites in aging brain tissues and the antiaging effect of the LSBG from the perspective of metabolic pathways based on LC-MS metabonomics technology;3.To study the anti-aging mechanism from the perspective of signaling pathways that related to metabolic pathways.Methods: 1.The aging model was used to evaluate the anti-aging efficacy of the LSBG.All rats were divided into 4 groups,control group,model group,low-doseage and high-dosage the LSBG(0.4g/kg and0.8g/kg).The behavioral tests were observed by using water maze test and open field test,and the effects of different dosage of LSBG on the cognitive abilities of aging rats were evaluated.Additionally,the pathological sections of hippocampus were observed.2.Metabolomics analysis of hippocampus and cortex tissues based on the LC-MS technology was performed to find potential biomarkers.These differential metabolites were further imported into the Metabo-Analyst database for pathway enrichment analysis to find metabolic pathways in brain that closely related to aging.3.Combined with literatures and the results of LC-MS metabolomics,the most relevant metabolic pathways were selected for verification.The kits were used to determine the content of metabolites and the activities of metabolic enzymes involved in key metabolic pathways of hippocampal and cortical samples.4.From the perspective of molecular biology,Western blotting was used to analyze the expression of key proteins in signaling pathways,which closely related to metabolic pathways,and to explore the regulate effect of the LSBG on the hippocampus and cortex tissues of D-gal-induced aging rat models.Results: 1.The results of the open field test and water maze test showed that the cognitive abilities of the aging rats that induced by D-gal was impaired.The learning and memory activities of aging rats were improved by the LSBG,and the improvement effect in the high dose was better than low dose.In addition,the LSBG could improve the neuron damage in hippocampus.2.The metabolomics results of hippocampus and cortex showed that there were 22 different metabolites in the cortex of aging rats.Among them,different doses the LSBG could regulate the content of 13 metabolites,involving in the biosynthesis of phenylalanine,tyrosine and tryptophan;glutamine-glutamate metabolism and so on.Meanwhile,21 different metabolites in hippocampus were disorder,and the LSBG could regulate the content of 14 metabolites,involving in the Alanine,Aspartic acid and Glutamate metabolism;Glutamine-Glutamine metabolism and so on.Glutamate metabolism were significantly changed both in hippocampus and cortex samples.In addition,as one of the excitatory neurotransmitters,glutamate played an important regulatory role in brain function and the development of the central nervous system.Therefore,glutamate metabolism was selected as the key pathway for verification in this study.3.Quantitative verification results of important metabolites in the glutamate metabolism showed that compared with the control group,the content of glutamate in the hippocampus(p<0.05)and cortex(p<0.001)tissues in the D-gal group rats were significantly increased.And the result was consistent with the LC-MS results,and the content of glutathione(p<0.05)in the two tissues was significantly reduced,it further indicated that the metabolism was disorders.The content of cysteine(p<0.01)in the cortex were significantly decreased.In this study,the content of glutamate,cysteine and glutathione in hippocampus and cortex tissues were regulated by the LSBG,the above results indicated that LSBG could effectively improve glutamate metabolism and glutathione synthesis.Further quantification of the key metabolic enzymes in the glutamate metabolism pathway,and the result showed that compared with the control group,the enzyme activities of glutaminase(GLS,p<0.01)and glutamine synthetase(GS,p<0.001)in the cortex of rats in the model group were significantly reduced,and it could influence glutamate-glutamine metabolism.In addition,the activities of glutamyl-cysteine-ligase(GCL)in the cortex(p<0.01)and hippocampus(p<0.05)tissues were significantly reduced,it suggested that glutamate synthesis and glutathione metabolism were disordered,and the oxidative defense system was disorder.And the activities of enzyme in hippocampus and cortex were regulated by the LSBG,it indicated that LSBG could affect glutamate metabolism and glutathione synthesis by improving the activities of metabolic enzymes.4.The results of western bolt analysis showed that compared with normal rats,the expression of Keap1 protein in hippocampus and cortex were significantly increased,and the expression of Nrf2,heme oxygenase(HO-1),quinone oxidoreductase 1(NQO-1),glutamylcysteine-ligase catalyze subunit GCLC were significantly decreased in aging rats.And the expressions of above proteins were regulated by LSBG.Conclusion: 1.The model of aging induced by D-gal could cause brain aging and impair the cognitive abilities of rats.And the cognitive function defects were improved by the LSBG.2.The metabolism disorder in hippocampus and cortex of aging rats were improved by the LSBG.3.The glutamate metabolism and its downstream glutathione metabolism could be regulated by the LSBG,and further the LSBG improved the oxidative stress.4.The expression of key proteins in Nrf2 signaling pathway were regulated by the LSBG. |
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