Protective Effect And Mechanism Of Lily Bulb And Rehmannia Decoction Medicated Serum On CORT-induced Nerve Cell Injury

Objective: To observe the protective effect of Lily Bulb and Rehmannia Decoction(LBRD)drug containing serum on nerve cell injury induced by corticosterone(CORT).To explore whether its mechanism is related to correcting the imbalance between excitatory and inhibitory neurotransmitter Glu and GABA.Methods: Firstly,UHPLC-Q-TOF/MS(LC-MS)was used to detect the components of LBRD drug-containing serum.Secondly,using the methods of serum pharmacology and nerve cell culture,Neuro-2a cells damaged by CORT were used to establish an vitro cell model suitable for studying the pathological mechanism of E:I imbalance and inflammatory injury in depression.Cell Counting Kit-8(CCK8)method was used to detect the effects of different concentrations of blank serum and drug-containing serum on the survival viability of normal culture Neuro-2a cells,so as to determine the appropriate serum working concentration.The cells were divided into control group,CORT model group and LBRD drug-containing serum group.The cells in the control group were cultured normally,while the model group was treated by CORT injury,and the LBRD group was treated by different concentrations of drug-containing serum on the basis of the model group.According to the results of CCK8 and lactate dehydrogenase(LDH),the optimal working concentration of LBRD drug-containing serum to protect CORT damaged cell model was determined.Elisa was used to detect the changes of neurotransmitter and inflammatory cytokines in control group,CORT model group and LBRD group.Hoechst33342 apoptosis staining was used to detect the apoptosis of each group.The protein expressions of VGAT and Gad67 were detected by Western Blot.The gene expressions of miRNA-144-3p,VGAT and Gad67 were detected by qRT-PCR.Finally,the expression of miRNA-144-3p was disturbed by transfection of pAV-CMV-GFP-miRNA-144-3p over-expressed plasmid and inhibitor expression plasmid to verify the effect of miRNA-144-3p on the gene expression of VGAT and Gad67 and GABA level.To comprehensively explore the molecular mechanism of LBRD protecting Neuro-2a cells from CORT damage and playing a neuroprotective role.Results:(1)Compared with the control cells,the cell survival rate decreased to 53%at the concentration of CORT 600 μM,which was determined to be the optimal concentration for CORT injury cell model(P<0.01).When the blank serum and LBRD drug-containing serum were lower than 20%,they had little effect on the survival rate of Neuro-2a cells,and were suitable as the pharmacological concentration range of drug-containing serum.(2)Compared with the control group,the cell viability of the model group was significantly decreased,and the LDH enzyme activity was significantly increased,and 1 h 10% LBRD drug-containing serum could improve the cell survival rate after CORT injury to the greatest extent(P<0.01).3.Compared with the control group,the levels of GABA,5-HT and IL-10 decreased and the levels of Glu and IL-1β increased after CORT injury.Compared with CORT model group,the levels of GABA,5-HT and IL-10 in LBRD drug-containing serum group were significantly increased(P<0.05,P<0.01),and the levels of Glu and IL-1β were decreased(P<0.01).These results indicate that the imbalance of neurotransmitter and excessive activation of inflammation in the CORT-induced cell model are consistent with the pathological mechanism of E:I imbalance and inflammatory injury in depression.The cell model is successfully constructed,and LBRD drug-containing serum can improve nerve cell damage by inhibiting inflammatory response and restoring neurotransmitter imbalance.(4)Hoechst 33342 apoptosis staining results showed that LBRD drug-containing serum could significantly increase the number,vitality and arrangement density of cells in the model group,reduce the degree of white plaque or crushed dense staining,and effectively inhibit cell apoptosis.(5)The results of Western Blot showed that LBRD drug-containing serum could significantly increase the protein expression of VGAT and Gad67 in the cell model(P<0.05).(6)The results of qRT-PCR showed that LBRD drug-containing serum could significantly reduce the expression of miRNA-144-3p and increase the mRNA expression of VGAT and Gad67 in the cell model(P<0.01).Disturbed expression of miRNA-144-3p can negative regulate the mRNA expression of Gad67 and VGAT,and affect the level of neurotransmitter GABA.Over-expression of miRNA-144-3p can antagonize the protective effect of LBRD drug-containing serum on CORT-induced Neuro-2a nerve cell damage.Conclusion: By increasing the level of anti-inflammatory factor IL-10,decreasing the level of pro-inflammatory factor IL-1β,increasing the level of GABA and 5-HT,and decreasing the level of Glu,LBRD drug-containing serum can restore the over-activation of inflammation and the imbalance of neurotransmitter induced by CORT.By increasing the expression of VGAT and Gad67 protein and gene,it can promote GABA synthesis and transport,increase GABA expression,restore E:I imbalance,and improve CORT-induced GABA damage in Neuro-2a nerve cells.miRNA-144-3p/Gad67/VGAT pathway is involved in the protective effect of LBRD drug-containing serum on CORT-induced GABA expression injury in nerve cells,which may be one of the important cellular mechanisms of the antidepressant effect of LBRD drug-containing serum.