| Background: Scutellaria baicalensis Georgi is a widely used perennial herb of the Lamiaceae,its root as a traditional medicinal part has the effects of clearing away heat and toxic material.As the non-medicinal parts of Scutellaria baicalensis Georgi,Scutellaria baicalensis stems and leaves are used as a different kind of tea,which has been edible for thousands of years.Recent pharmacological studies have shown that Scutellaria baicalensis leaves also have extensive and powerful effects,including anti-oxidation,liver protection,anti-aging and improvement of memory deficits.In recent years,our laboratory has conducted so much research on the anti-aging efficacy of Scutellaria baicalensis leaves.Studies showed that Scutellaria baicalensis leaves have anti-aging effects in different animal models included rats and fruit flies.However,the research on the anti-aging mechanism of Scutellaria baicalensis leaves is still not thorough enough.Therefore,based on the theoretical basis that the liver plays an important role in the aging process,this paper will conduct in-depth research on the anti-aging mechanism of Scutellaria baicalensis Leaves by non-targeted liver metabolomics,combined with targeted quantitative analysis and molecular biology methods,to lay a foundation for the deep utilization and resource development of Scutellaria baicalensis Georgi.Objective: 1.To explore the regulation mechanism of Scutellaria Baicalensis Leaves extract(SLE)on liver metabolic profile of D-Galactose(D-gal)-induced aging rats based on liver non-targeted metabolomics,then screened out the targeted metabolic pathways related to the aging process;2.Using targeted quantitative analysis methods to quantitatively verify the key metabolic pathways in the liver of D-gal-induced aging rats by SLE;3.Based on the results of metabolomics,using molecular Biological technology to explore the effect of SLE on the Nrf2/Keap1 pathway in the liver of D-gal-induced aging rats.Methods: 1.Based on UPLC-MS/MS metabolomics technology,liver from D-gal-induced aging rat was subjected to metabolic profile analysis,and differential metabolites related to aging were screened by multivariate statistical methods and analysis of metabolic pathways were imported into the metaboanalyst;2.Based on the results of non-targeted metabolomics,UPLC-MS/MS and kits were applied for targeted quantitative analysis of metabolites in the pathway;3.Use kits to determine the activity of key enzymes in the hepatic metabolic pathway,including γ-glutamyl cysteine ligase(GCL),Glutamine synthetase(GS),and Glutaminase(GLS),Glutathione reductase(GR),glutathione S-transferase(GST);and measure the level of malondialdehyde(MDA)in liver tissue,superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)activity.4.Using Western blot to explore the effect of SLE on the expression of Nrf2/Keap1 pathway in the D-gal aging model.Results: 1.A total of 39 liver metabolites related to aging and SLE regulated were identified by non-targeted metabolomics technology,mainly involved the metabolism of glutamine and glutamate,the biosynthesis of phenylalanine,tyrosine and tryptophan,and the metabolism of alanine,aspartic acid and glutamate,etc.,of which glutamine and glutamate metabolism were the most important.This result can be used to corroborate and improve the results of metabolomics.2.The results of targeted quantitative analysis showed that the level of glutamate(Glu),glutamine(Gln),reduced glutathione(GSH)and oxidized glutathione(GSSG)in the liver of D-gal group rats decreased significantly(P<0.05),but the content of cystine(Cys)and methionine(Met)has a tendency to decrease;After administration of SLE,the levels of glutamate,glutamine,GSH and GSSG in the liver of aging rats increased significantly.3.The key enzyme assay results showed that SLE can reverse the significantly decrease of glutamine synthetase(GS),γ-glutamyl cysteine ligase(GCL)and glutathione S-transfer(GST)activities in the liver of D-gal-induced aging rats and a decrease trend of glutathione reductase(GR),but have no significant effect on glutaminase(GLS).Antioxidant index results showed that SLE can reverse the increase of MDA content in the liver of aging rats,the decrease of SOD and GSH-Px activity,improve oxidative stress damage,and have obvious antioxidant effects.4.Western blot results showed that the protein expression of Nrf2,GCLC,GCLM,NQO1 and HO-1 in the liver of aging rats was reduced,while the protein expression of Keap1 was increased;After administration of SLE,the protein expression of Nrf2,GCLC,GCLM,NQO1 and HO-1upregulated,and the protein expression of Keap1 downregulated.Conclusion: SLE can significantly improve the abnormal liver metabolism of aging rats induced by D-gal to delay aging,which regulated liver glutamine-glutamate metabolism and glutathione synthesis metabolism,and activated Nrf2/ Keap1 signaling pathway proteins were closely related. |
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