The Effects And Mechanisms Of Baicalein In Inhibiting H₂O₂-induced Astrocyte Senescence Based On Senescence-associated Secretory Phenotype（SASP）

Scutellaria baicalensis Georgi belongs to the Lamiaceae plant,and baicalein is an active flavonoid component extracted from the roots of Scutellaria baicalensis Georgi.It has been demonstrated that baicalein could improve memory impairment in multiple animal models,however,the underlying mechanisms remain ambiguous.Aging is the main risk factor for memory impairment.The abnormal accumulation of senescent cells in the brain and the secretion of senescence-associated secretory phenotype（SASP）are closely related to neurodegeneration and cognitive impairment.Therefore,this study explored the protective effects and the anti-senescence effects of baicalein in the H₂O₂-induced senescence model of astrocytes,as well as its molecular mechanisms and metabolic mechanisms.Our results demonstrated that treatment with baicalein can delay H₂O₂-induced astrocyte senescence by reducing the activity of SA-β-gal,reducing the number of cells in the S phase,and down-regulating the levels of p16-INK4A and p21 proteins.Baicalein protects astrocytes from H₂O₂ induced damage through elevation of cell viability,improvement of cell morphology,increasing cloning and colony formation,and reducing oxidative damage（MDA and LDH levels）.Baicalein can inhibit the secretion of SASP（IL-6,IL-8,TNF-α,CXCL1 and MMP-1）and the expression of mRNA,and inhibit NF-κB and JAK2/STAT1 pathways which are related to SASP.The results of ¹H-NMR metabolomics study showed that baicalein can regulate 10 different metabolites including leucine and threonine,involved in 5metabolic pathways including arginine biosynthesis.The correlation analysis between differential metabolites and SASP showed that leucine is significantly correlated with SASP.Further study demonstrated that supplement with leucine could restrain SASP secretion,and baicalein could significantly increase leucine level through down-regulation of BCAT1 and up-regulation of SLC7A5 expression.In summary,baicalein exerted protective and anti-senescence effects in H₂O₂-induced senescent astrocytes possibly through inhibition of SASP,suppression of JAK2/STAT1/NF-κB pathway,and regulation of leucine metabolism,providing new ideas for the treatment of neurodegenerative diseases such as Alzheimer’s disease.Objective:The H₂O₂-induced T98G astrocyte senescence model was used to verify the protective and anti-aging effects of baicalein in H₂O₂-induced senescent astrocytes,as well as the suppression of senescence-related secretory phenotype（SASP）;Clarify the molecular and metabolic mechanisms of baicalein in delaying the senescence of astrocytes.Methods:1.200μM H₂O₂ was used to induce senescence and damage of T98G cells,and to evaluate the effect of baicalein in delaying the senescence of T98G cells induced by H₂O₂ by detecting senescence markers SA-β-gal,cell cycle,p16-INK4A and p21;Cell viability,cell clone formation,MDA and LDH assays were conducted to investigate the protective effect of baicalein on H₂O₂-induced T98G cells damage.2.The ELISA method was used to evaluate the inhibition effect of baicalein on SASP secreted by H₂O₂-induced T98G cells;q RT-PCR method was used to investigate the effect of baicalein on mRNA levels of SASP in H₂O₂-induced senescent astrocytes,and Western Blot was used to investigate the regulator effect of baicalein on NF-κB,p-NF-κB,JAK2,p-JAK2,STAT1 and p-STAT1 expression in H₂O₂-induced senescent astrocytes.3.¹H-NMR metabonomics technology was used to find the differential metabolites and metabolic pathways regulated by baicalein,the metabolites related to SASP were found through correlation analysis,ELISA method was used to detect the effect of leucine on leucine level and SASP secretion in H₂O₂-induced T98G cells,and to investigate the effect of baicalein on intracellular leucine level and the key enzymes（leucine aminotransferase,leucine dehydrogenase and branched chain amino acid transferase）that regulate leucine.Western blot technology was used to investigate the regulation effect of baicalein on leucine transporter SLC7A5 to study the mechanism of baicalein in regulating leucine metabolism.Results:1.T98G cells exposed to 200μM H₂O₂for 8 hours can successfully establish the senescence model and damage model.Pretreatment with baicalein for 2 h can significantly reduce the activity of SA-β-gal enzyme,reduce the number of cells in S phase,and down-regulate the expression of p16-INK4A and p21,indicating that baicalein can delay cell senescence.In addition,baicalein can significantly increase cell viability,improve cell morphology,increase cell cloning and colony formation,reduce extracellular LDH levels and intracellular MDA levels,indicating that baicalein can protect from H₂O₂-induced cell damage.2.Baicalein can inhibit SASP（IL-6,IL-8,TNF-α,CXCL1 and MMP1）secretion in H₂O₂-induced senescent astrocyte,and down-regulate mRNA expression of SASP,promote the secretion of HGF and increase its mRNA expression.Meanwhile,baicalein can restrain NF-κB and JAK2/STAT1 pathway.The results indicated that baicalein can regulate the secretion of SASP and its mRNA levels,and regulate SASP-related pathways NF-κB and JAK2/STAT1.3.¹H-NMR metabolomics analysis results showed that baicalein significantly up-regulates 10 differential metabolites,involved in 5 metabolic pathways,including arginine biosynthesis,arginine and proline metabolism.The correlation analysis between the differential metabolites and SASP showed that leucine was significantly related to the 6 SASP factors.Further research found that supplementation with leucine（400-600μM）can inhibit the secretion of SASP（IL-8,TNF-αand CXCL1）.Baicalein may promote the accumulation of leucine in cells through down-regulation of BCAT1and up-regulation of SLC7A5.Conclusion:Baicalein delays H₂O₂-induced astrocytes senescence through inhibition of senescence-associated secretory phenotype（SASP）,suppression of JAK2/STAT1/NF-κB pathway and regulation of leucine metabolism.