Protective Effects And Mechanisms Of Baicalein On Neuroinflammation Of LPS-activated BV-2 Cells

Rationales: Aging is a phenomenon accompanied by decline in multiple tissues and organs with age.The central nervous system is one of the systems most affected by aging.Some characteristic changes in the aging brain induce neurodegenerative diseases and aging cognitive dysfunction,seriously affecting the physical and mental health,and life quality of the elderly.Low immunity,metabolic disorders and oxidative stress can cause inflammatory response in central nervous system of the elderly,which in turn exacerbates the occurrence and development of neurodegenerative diseases and aging cognitive dysfunction.However,there are no clinically available drugs for improving or reversing aging cognitive dysfunction.Our previous research has shown that baicalein can improve cognitive dysfunction in two aging animal models,including D-galactose-induced aging rats and senescence accelerated mouse prone 8?SAMP8?.However,the mechanism of baicalein on improving senile cognitive dysfunction is unclear.Since the brain aging process is accompanied by the generation of neuroinflammation,therefore this project explores the protective effect and mechanism of baicalein on neuroinflammation in lipopolysaccharide?LPS?-activated BV-2 microglia cells.Objective: The model of LPS-activated BV-2 cells was used to verify the neuroprotective effects of baicalein;to explore the mechanism of baicalein on anti-neuroinflammation and anti-oxidative stress in LPS-activated BV-2 cells;to clarify the regulation mechanism of baicalein on metabolism abnormality in LPS-activated BV-2 cells.Methods: 1.Cell morphology observation,cell viability measurement and NO production measurement were used to verify the establishment of the LPS-activated BV-2 cell model.In order to evaluate the neuroprotective effect of baicalein,the effects of baicalein on cell viability and release of pro-inflammatory factors were detected in LPS-activated BV-2 cells.2.Western blot and immunofluorescence were used to investigate the molecular mechanism of baicalein on anti-neuroinflammation and anti-oxidative stress in LPS-activated BV-2 cells,and associated proteins in key pathway were verified.3.¹H-NMR metabolomics analysis approach was used to find differential metabolites and analyze metabolic pathways.The differential metabolites regulated by baicalein were obtained,and the molecular mechanism of baicalein on regulating metabolic abnormalities was elucidated.Results:1.According to the experimental results of cell morphology,cell viability and NO production,the final modeling conditions were determined by serum-free medium for 6 h and 0.1 ?g/m L LPS treatment for 24 h,which used for evaluating the neuroprotective effect of baicalein.The results showed that 0.5,1,2and 4 ?M baicalein had no effect on the cell viability in LPS-activated BV-2 cells,however,0.5,1,2 and 4 ?M baicalein significantly inhibited NO,IL-6 and TNF-?levels in culture supernatant in LPS-activated BV-2 cells with a dose-dependent manner.2.In the LPS-activated BV-2 cells,different concentrations of baicalein could significantly inhibit ROS production.4 ?M baicalin significantly inhibited the expression of inflammatory pathway-related proteins,including iNOS,COX-2 and NF-?B/p65,and inhibited the phosphorylation of NF-?B and STAT1.In addition,baicalein significantly inhibited the expression of membrane p47^phox protein,total gp91^phox protein and membrane gp91^phox protein in LPS-activated BV-2 cells.The results indicated that the effect of baicalein on anti-neuroinflammation and anti-oxidative stress in LPS-activated BV-2 cells was related to inhibition of NOX2/STAT1/NF-?B signaling pathway.3.1H-NMR metabolomics study showed that baicalein can reversely regulate 12 differential metabolites in LPS-activated BV-2 cells,which involved in four metabolic pathways,including alanine,aspartate and glutamate metabolism,glutathione metabolism,arginine and proline metabolism,D-glutamine and D-glutamate metabolism.Conclusion: Baicalein has protective effects on neuroinflammation in LPS-activated BV-2 cells,and its mechanism was closely related to the inhibition of NOX2/STAT1/NF-?B signaling pathway and regulation of metabolic abnormalities.