| ObjectiveNearly 50 million people worldwide suffer from Alzheimer’s disease(AD)or ADrelated dementia,and China has the largest and fastest-growing number of AD patients in the world.But there are currently no drugs that can stop the progression of the disease,and the development of therapies and drugs for AD is urgent.Traditional Chinese medicine is highly effective in the prevention and treatment of AD.The study of the combination of active compounds in traditional Chinese medicines is an important method for studying the material basis and the mechanism of action of Chinese medicine compounding.Paeoniflorin(PF),Ferulic acid(FA),and Atractylenolide III(ATL)are the active monomeric components of Paeonia lactiflora Pall(Paeoniaceae),Angelica sinensis(Oliv.)Diels(Umbelliferae),and Atractylodes.macrocephala Koidz.(Asteraceae),respectively.In this experiment,three monomers,PF,FA,and ATL,were selected to study their effects on microglia inflammation and autophagy alone and in combination,to elucidate the possible molecular mechanisms of the combination of PF,FA,and ATL monomers for the treatment of AD,and to provide some theoretical basis for the study of the combination of active ingredients in traditional Chinese medicine.Methods1.Effects of PF,FA,and ATL alone and in combination on LPS-induced inflammation in BV2 cells1 μg/mL LPS was used to induce BV2 cells to establish a neuroinflammation model;MTT method was used to screen the concentration of PF,FA and ATL administered alone and in combination;ELISA was used to detect IL-1β,IL-6 and TNF-α concentrations in cell supernatants;RT-qPCR was used to detect IL-1β,IL-6 and TNF-α mRNA expression;IL-1βand TNF-α protein expression was detect by Western blot.This was used to observe whether PF,FA,and ATL alone and in combination had anti-inflammatory effects and whether the effect of the combination was different from that of single use.2.Effects of PF,FA,and ATL alone and in combination on the autophagy proteins LC3 and p62 of LPS-induced BV2 cellsWestern blot and immunocellulerchemistry were used to detect LC3 and p62 protein expression.Observed whether PF,FA and ATL alone and in combination used affected LPSinduced autophagy in BV2 cells.3.Study on the mechanism of anti-inflammatory effect of PF,FA,and ATL in combination used on LPS-induced BV2 cellsAfter pretreatment with autophagy inhibitor wortmannin(Wor),the effect of PF,FA,and ATL in combination used on IL-1β and IL-6 mRNA expression was observed by RTqPCR,and IL-6 protein expression was detected by Western blot,thus observing the effect of autophagy inhibitor on the anti-inflammatory effect of PF,FA,and ATL in combination.After pretreatment with the autophagy inhibitor Wor and the autophagy agonist rapamycin(Rapa),the expression of autophagy-related proteins p-AMPK,p-ULK1,Beclinl,LC3,and p62 proteins in BV2 cells was detected by Western blot,and the localization and expression of LC3 and p62 proteins were observed by immunocellulerchemistry,thereby observing the regulation of autophagy pathway proteins in LPS-induced B V2 cells by the combination used of PF,FA,and ATL.The effect of PF,FA and ATL in combination used on TFEB and LAMP2A expression was observed by Western blot after pretreatment with the autophagy inhibitor Wor,thus observing whether PF,FA,and ATL in combination used can regulate TFEB and LAMP2A expression.Results1.Effects of PF,FA,and ATL alone and in combination on LPS-induced inflammation in BV2 cells.(1)After 1 μg/mL LPS induced BV2 for 12 h,IL-1β,IL-6,and TNF-α mRNA expression were significantly increased(P<0.01),this modeling condition was used for subsequent experiments.MTT results showed no effect of 15 μM PF,55 μM FA and 35 μM ATL on cell viability either alone or in combination,and this concentration was selected for subsequent experiments.(2)ELISA results showed that IL-1β,IL-6 and TNF-α concentrations were significantly higher in the LPS group compared with the normal control group(P<0.01),and PF,FA and ATL administration reduced IL-1β,IL-6 and TNF-α expression to different degrees.The PF+FA+ATL group had the most significant reduction effect of IL-1β and IL-6,and the difference was statistically significant compared with each single group and two-two combination groups(P<0.05).(3)RT-qPCR results showed that IL-1β,IL-6,TNF-α mRNA expression was significantly higher in the LPS group compared with the normal control group(P<0.01),and PF,FA,and ATL administration could reduce IL-1β,IL-6,TNF-α mRNA expression to different degrees(P<0.05).For IL-1β and IL-6 mRNA inhibition,the effect of the PF+ATL and FA+ATL group was better than that of the alone used group(P<0.05),and the effect of PF+FA+ATL group was stronger than that of alone used group and PF+ATL,FA+ATL group(P<0.05).(4)Western blot results showed that PF,FA and ATL administration either alone or in combination decreased IL-1β and TNF-α protein expression(P<0.05).2.Effects of PF,FA,and ATL alone and in combination on the autophagy proteins LC3 and p62 of LPS-induced BV2 cellsThe effects of PF,FA,and ATL alone and in combination on LPS-induced autophagic proteins LC3 and p62 in BV2 cells were observed by Western blot and immunocellulerchemistry.The results showed that LC3 protein expression was significantly lower(P<0.01)and p62 protein expression was significantly higher(P<0.01)after LPS induction compared with normal control group.the administration of PF,FA and ATL increased LC3 protein expression(P<0.01)and decreased p62 protein expression(P<0.05),and the combination group of PF,FA,and ATL had better effects on LC3 and p62 regulation than each single group.3.Study on the mechanism of anti-inflammatory effect of PF,FA and ATL in combination used on LPS-induced BV2 cells(1)Western blot results showed that p-AMPK,p-ULK1,Beclinl,LC3 protein expression was significantly lower(P<0.01,P<0.01,P<0.05,P<0.01)and p62 protein expression was significantly higher(P<0.01)in BV2 cells after LPS induction compared to normal control group.p-AMPK,p-ULK1,Beclinl,LC3 protein expression was significantly increased(P<0.01,P<0.01,P<0.01,P<0.01)and p62 protein expression was significantly decreased(P<0.01)after the PF,FA,ATL in combination used and Rapa administration.(2)RT-qPCR results showed that IL-1β and IL-6 mRNA expression was significantly higher in BV2 cells after LPS induction compared with normal control(P<0.05),and IL-1βand IL-6 mRNA expression was significantly lower after PF,FA,and ATL coadministration(P<0.05),while after adding the inhibitor Wor,compared with PF+FA+ATL group,IL-1βand IL-6 mRNA expression was significantly higher in Wor group(P<0.05).Western blot results showed that IL-6 protein expression was significantly increased after LPS induction(P<0.01),and IL-6 was significantly decreased after PF,FA,and ATL co-administration(p<0.05),while IL-6 expression was significantly increased after the addition of inhibitor Wor(P<0.05).(3)Western blot results showed that after pretreatment with the addition of autophagy inhibitor Wor,p-AMPK,p-ULK1,Beclinl,LC3 protein expression was significantly lower(P<0.05,P<0.01,P<0.01,P<0.05)and p62 protein expression was significantly higher in the Wor group compared with the PF+FA+ATL group(P<0.01).In addition,the expression of LC3 and p62 was detected by immunocellulerchemistry,and the results were consistent with the Western blot results.(4)Compared with the normal control group,TFEB and LAMP2A protein expression in BV2 cells was significantly decreased after LPS induction(P<0.01,P<0.01),and TFEB and LAMP2A protein expression were significantly increased after PF,FA,and ATL coadministration compared with the LPS model group(P<0.05,P<0.05),while after adding the inhibitor Wor,TFEB and LAMP2A protein expression was significantly reduced(P<0.01,P<0.05).ConclusionIn LPS-induced BV2 cells,the active components PF,FA,and ATL can inhibit the expression of IL-1β,IL-6 and TNF-α and attenuate the inflammatory response of BV2 cells,and the combination of PF,FA,and ATL may have synergistic effects.The anti-inflammatory effect of PF,FA and ATL combination was closely related to their activation of AMPK/ULK1/Beclinl pathway and regulation of TFEB and LAMP2A expression. |
PDF Full Text Request