The Research Of Cannabidiol Through PPAR-γ Receptors Attenuating The Activation Of NLRP3 Inflammasomes In BV2 Cells By Methamphetamine

Objective:Methamphetamine(METH)is the secondly most widely abused drug in the world except cannabis.Because of its damage to the central nervous system,it endangers human health and social public safety seriously.The role of neuroinflammation has been attracted much attention in the mechanism of damage of METH to the central nervous system.Studies have shown that Nod-like receptor protein 3(NLRP3)inflammasomes play important roles in METH-mediated neurotoxicity,but its mechanism is still unclear.Cannabidiol(CBD)is one of the main non-psychoactive compounds in cannabis plants.It has high tolerance,extensive anti-inflammatory and neuroprotective pharmacological effects in human and animal experiments.In addition,peroxisome proliferator-activated receptor(PPAR)-y plays an anti-inflammatory effect in neurodegenerative diseases,which may also be one of the potential targets of CBD.However,whether CBD attenuates the activation of NLRP3 inflammasomes in BV2 cells by METH through PPAR-y receptors has not been reported.Therefore,this study intends to observe the role and intervention mechanism of CBD in the activation of NLRP3 inflammasomes in BV2 cells by METH,that aims to reveal the neurotoxicity mechanism of METH and provide a theoretical basis for the treatment of METH abuse.Methods:Immunofluorescence,Western blot,IL-1β,IL-18 and CCK-8 detection kits were used.In the experiments,microglia(BV2 cells)were selected as experimental objects.Steps:(1)BV2 cells were treated with different concentrations of METH(0.025,0.05,0.1,0.2,0.4,and 0.8 mM)for 12 h,and selected the best METH concentration to treat BV2 cells for 0,0.5,1,3,6,12,and 24 h.The expression of NLRP3,Caspase-1 and ASC proteins in BV2 cells in each group were detected by Western blot(WB)to determine the optimal concentration and time point for METH to activate NLRP3 inflammasomes.(2)BV2 cells were pretreated with different concentrations of CBD(1uM,10uM)for 1 hour.After 12h,the expression of NLRP3,Caspase-1,ASC,GSDMD,GSDMD-N,pro-inflammatory factor IL-1β and IL-18 proteins in BV2 cells of each group were detected by WB.(3)BV2 cells were treated with different concentrations of METH for different time,then the expression of PPAR-γ protein were detected.After BV2 cells were pretreated with PPAR-γ antagonist(GW9662)and CBD,the expression of proteins such as inflammasomes,IL-1β,IL-18,PPAR-γ proteins in BV2 cells were detected.by WB and immunofluorescence.The cell activity of each group were also detected by CCK-8 method.Results:1 METH activates NLRP3 inflammasomes in BV2 cellsThe expression of NLRP3,ASC and Caspase-1 proteins increased with the increase of METH concentration.After BV2 cells were treated with 0.1 mM METH for different time,the expression of NLRP3,ASC and Caspase-1 proteins gradually increased as the time of METH administration increased.2.CBD inhibits the activation of NLRP3 inflammasomes in BV2 cells by METHCompared with the control group,the expression of NLRP3,Caspase-1,ASC,GSDMD,GSDMD-N,IL-1β and IL-18 proteins in the METH group were significantly increased.Using CBD intervention in advance,the expression of those proteins were significantly reduced.These results indicate that CBD can inhibit the activation of NLRP3 inflammasomes in BV2 cells by METH.3.METH activates the NLRP3 inflammasomes of BV2 cells via PPAR-γ pathwayBV2 cells were treated with different concentrations of METH for 12 hours,the expression level of PPAR-y protein gradually decreased with the increase of the concentration of administration.Treatment of BV2 cells with 0.1 mM METH for different times,the expression level of PPAR-γ protein gradually was decreased.Effect of METH on PPAR-y protein expression was concentration-and time-dependent.Intervention with PPAR-y-specific antagonist GW9662 can increase the expression levels of NLRP3,Caspase-1,ASC,GSDMD,GSDMD-N,IL-1β and IL-18 proteins in BV2 cells induced by METH.The results indicate that METH activates the NLRP3 inflammasomes of BV2 cells via PPAR-γ pathway.4.CBD inhibits the activation of NLRP3 inflammasome in BV2 cells by METH via PPAR-γ pathwayThe expression level of PPAR-γ protein increased in a concentration-dependent manner,after BV2 cells were treated with 0.1 uM,1uM and 10uM CBD.The result shows that CBD can increase the expression of PPAR-γ protein.Pretreatment with CBD for 1 hour,the expression of NLRP3,Caspase-1,ASC,GSDMD,GSDMD-N,IL-1β,IL-18 proteins and the concentration of IL-1β and IL-18 in the supernatant of BV2 cells were increased in the CBD10uM+METH+GW9662 group,compared to CBD10uM+METH group.Moreover the intensity of PPAR-γ immunofluorescence was weakened and the survival rate of BV2 cells was decreased.Conclusions:1.METH can activate NLRP3 inflammasomes in BV2 cells.2.CBD inhibits the activation of NLRP3 inflammasomes in BV2 cells by METH via PPAR-γ pathway.