Study On Gastrodin Alleviated Methamphetamine-induced Neuroinflammation Through TLR4/MyD88/NF-κB Pathway

Objective:To clarify the relationship between neuroinflammation caused by methamphetamine(MA)and TLR4/My D88/NF-κ B signal pathway.To investigate the role of gastrodin which could regulate the microglia cell inflammation induced by MA through TLR4/My D88/NF-κB signaling pathway.And then,to clarify whether gastrodin has a protective effect on MA induced neurotoxicity,and the potential mechanism anti-inflammatory effects of gastrodin.Methods:1.BV2 microglia were cultured with methamphetamine administration(1)The model of MA induced microglia inflammation was established.BV2 cells were treated with 300 μM and 600 μM MA for 24 h,and the changes of inflammatory cytokines,which were IL-18 and IL-1β,were detected by ELISA kit.(2)After intervention with gastrodin,the changes of inflammatory factors were detected.ELISA kit and Western blot were used to detect the changes of inflammatory cytokines IL-18 and IL-1β.(3)The effect of TLR4/My D88/NF-κB signaling pathway on microglia cell inflammation which induced by MA.After TLR4 inhibitor TAK-242 was used or TLR4 was silenced by lentivirus,the changes of inflammatory factors,apoptosis-related protein and TLR4/My D88/NF-κB signaling pathway were detected.Western blot technique was used to detect the changes of inflammatory cytokines IL-18 and IL-1β,apoptosis-related protein Bax,Bcl-2 and key signaling molecules TLR4,My D88,NF-κBp65,and p-NF-κBp65 in TLR4/My D88/NF-κB pathway.(4)The effect of TLR4/My D88/NF-κB signaling pathway on gastrodin against microglia cell inflammation which induced by MA.After intervention with gastrodin,the changes of inflammatory factors,apoptosis-related prote After intervention with gastrodin,in and TLR4/My D88/NF-κB signaling pathway were detected.Western blot technique was used to detect the changes of inflammatory cytokines IL-18 and IL-1β,apoptosis-related protein Bcl-2,Bax and key signaling molecules TLR4,My D88 and p-NF-κBp65 in TLR4/My D88/NF-κB pathway.2.Primary microglia of SD rat brain samples were cultured with methamphetamine administration s(1)After gastrodin intervention,the changes of inflammatory factors were detected.ELISA kit and Western blot were used to detect the changes of inflammatory cytokines IL-18 and IL-1β.(2)The effect of TLR4/My D88/NF-κB signaling pathway on microglia cell inflammation which induced by MA.After TLR4 gene was inhibited by inhibitor,TAK-242,the changes of inflammatory factors,apoptotic-related protein and key signaling molecules of TLR4/My D88/NF-κB signaling pathway were detected.Western blot technique was used to detect the changes of inflammatory cytokines IL-18 and IL-1β,apoptosis-related protein Bax,Bcl-2 and key signaling molecules TLR4,My D88,NF-κBp65 and p-NF-κBp65 in TLR4/My D88/NF-κB pathway.(3)The effect of TLR4/My D88/NF-κB signaling pathway on gastrodin against microglia cell inflammation which induced by MA.After intervention with gastrodin,the changes of inflammatory factors,apoptosis-related protein and TLR4/My D88/NF-κB signaling pathway were detected.Western blot technique was used to detect the changes of inflammatory cytokines IL-18 and IL-1β,apoptosis-related protein Bcl-2,Bax and key signaling molecules TLR4,My D88 and p-NF-κBp65 in TLR4/My D88/NF-κB pathway.Results:1.MA induced inflammation in microglia cells,and the expressions of IL-18 and IL-1β increased significantly.2.Gastrodin could reduce the neuroinflammation induced by MA,and the expressions of IL-18 and IL-1β were significantly decreased.3.MA could induce neuroinflammation in microglia through TLR4/My D88/NF-κB signaling pathway.(1)After intervention with TLR4 inhibitor TAK-242,compared with the control group,the expressions of inflammatory cytokines IL-18 and IL-1β were significantly increased in MA group,while apoptosis-related protein Bcl-2/Bax had no significant changes.The expressions of key signaling molecules of TLR4/My D88/NF-κB pathway,TLR4,My D88 and p-NF-κBp65 were also significantly increased,while NF-κBP65 was no significant change,but p-NF-κBp65/ NF-κB was significantly increased.After TAK-242 intervention,Compared with the MA group,the expressions of inflammatory cytokines IL-18 and IL-1β were decreased significantly,while apoptosis-related protein Bcl-2/Bax had no significant change,the expressions of TLR4,My D88 and p-NF-κBp65 were decreased significantly,while NF-κBp65remained had no significant change.However,the ratio p-NF-κBp65/ NF-κBp65 was significantly reduced.(2)After TLR4 was silenced by lentivirus,compared with the control group,the expressions of the inflammatory cytokines IL-18 and IL-1β were significantly increased in MA group,while the apoptosis-related protein Bcl-2/Bax had no significant change.The expressions of TLR4,My D88 and p-NF-κBp65 was also significantly increased,while the NF-κBp65 was unchanged.However,the ratio p-NF-κBp65/ NF-κBp65 increased significantly.Compared with the MA group,After TLR4 was silenced by lentivirus,the expressions of the inflammatory factors IL-18 and IL-1β were significantly decreased,while the apoptosis-related protein Bcl-2/Bax had no significant changes.The expressions of TLR4,My D88 and p-NF-κBp65 were significantly decreased,while NF-κBp65 was unchanged,the ratio p-NF-κBp65/NF-κBp65 was significantly reduced.4.Gastrodin could reduce methamphetamine-induced microglia inflammation through TLR4/My D88/NF-κB signaling pathway.Compared with the control group,the expressions of inflammatory cytokines IL-18 and IL-1β were significantly increased in MA group,while apoptosis-related protein Bcl-2/Bax had no significant change.the expressions of TLR4,My D88,and p NF-κBp65 also significantly increased.Compared with the MA group,the expressions of the inflammatory factors IL-18 and IL-1β were significantly decreased after the intervention with gastrodin,while the apoptosis-related protein Bcl-2/Bax had no significant change.The expressions of TLR4,My D88,and p-NF-κB were significantly decreased.Conclusions:1.Methamphetamine induced inflammation in microglia.2.Microglia inflammation which induced by methamphetamine was related to the TLR4/My D88/NF-κB signaling pathway.3.Gastrodin could alleviate methamphetamine-induced microglia inflammation through regulating TLR4/My D88/NF-κB signaling pathway.