Effect Of Gastrodin On TLR4/MyD88/NF-κB Signal Pathway In Hippocampus Of Methamphetamine Dependent CPP Rats

Objective:1.To establish a methamphetamine-dependent conditioned place preference(CPP)rat model,to detect the protein expressions of inflammatory factors IL-1 β and TNF-a in the hippocampus,to detect the protein and mRNA expressions of key factors in TLR4/MyD88/NF-κ B signal pathway,to explore the relationship between hippocampus neuroinflammation and TLR4/MyD88/NF-κ B signal pathway in Meth-dependent CPP rats.2.Intervention with gastrodin(Gas)and then detect the morphological changes of microglia,the protein expressions of inflammatory factors IL-1 β and TNF-α,and the protein and mRNA expressions of key factors in TLR4/MyD88/NF-κB signal pathway in the hippocampus of Meth-dependent CPP rats after gastrodin intervention,and to explore the effects of gastrodin on hippocampus neuritis and TLR4/MyD88/NF-κB signal pathway in Meth-dependent CPP rats.3.Methods 1.80 SD male rats were randomly divided into 8 groups(n=10),which were saline group,Meth group,TAK-242 group,Meth+TAK-242 group,Meth+Gas 10mg/kg group,Meth+Gas 30mg/kg group,Meth+Gas 100mg/kg group,and Gas 100mg/kg group.The method of administration TAK-242 group and METH+TAK-242 group were intraperitoneally injected with TAK-242(3mg/kg),and the Meth+TAK-242 group was injected with METH(10mg/kg)after 1 hour.Saline group and Gas100 mg/kg group were injected intraperitoneally with normal saline(lOmg/kg)and gastrodin(100mg/kg)respectively.Meth+Gas 10mg/kg group,Meth+Gas 30mg/kg group and Meth+Gas 100mg/kg group were injected intraperitoneally with Meth(10mg/kg),all the rats in 8 groups were given drugs for continuous 14 days.Then the conditioned place preference(CPP)test was used for determining the dependence of rats.Then the rats in Meth+Gas10mg/kg group,Meth+Gas30mg/kg group,and Meth+Gas100mg/kg group were treated with gastrodin(10mg/kg,30mg/kg,or 100mg/kg)for 14 days,respectively.The conditioned place preference(CPP)test was used to detect the effect of gastrodin on Meth-induced CPP rat model.4.Immunofluorescence technique was used to detect the morphological changes of microglia,Iba-1 was as a microglial marker in the hippocampus of Meth-dependent CPP rats;Western Blot was used to detect the protein expressions of IL-1 β,TNF-α,TLR4,MyD88,TRAF6,iκB-α,p-iκB-α,NF-κBp65,and p-NF-κBp65 in the hippocampus of Meth-dependent CPP rats;Fluorescence quantitative PCR was used to detect the mRNA expressions of TLR4,MyD88,TRAF6,iκB-α,and NF-κBp65 in the hippocampus of Meth dependent CPP rats.Results1.The rat model of Meth dependence was established successfully.Compared with the saline group,the time of the rats stayed in the drug-paired chamber of Meth group was significantly longer than that in the saline group(P<0.01),which suggested that the CPP model of Meth dependent rats was established successfully.2.The expression of inflammatory factors IL-1β,TNF-α and key factors in TLR4/MyD88/NF-κB pathway in the hippocampus of Meth-dependent CPP rats.Compared with saline group,the protein and mRNA expressions of TLR4,MyD88,TRAF6,and NF-κBp65 increased(P<0.001 or P<0.01),The expressions of protein and mRNA of iκB-α were decreased(P<0.01),The protein expressions of p-iκB-α,p-NF-κBp65,IL-1β,and TNF-α were increased in Meth group(P<0.01 or P<0.05);Compared with Meth group,the protein and mRNA expressions of TLR4,MyD88,TRAF6,and NF-κBp65 were decreased,the protein and mRNA expressions of IκB-αincreased,the protein expression of p-iκB-α,p-NF-κBp65,IL-1β,and TNF-αdecreased in Meth+TAK-242 group(P<0.001 or P<0.05).3.The effect of different gastrodin on the CPP effect of Meth-dependent rats.Compared with the Meth group,10 mg/kg,30 mg/kg,and 100 mg/kg gastrodin could reduce the Meth-induced CPP effect in a dose-dependent manner(P<0.01),the CPP effect had a dose-effect relationship with gastrodin.4.Effects of different doses of gastrodin on the activation of hippocampal microglia in Meth-dependent CPP rats.Compared with the saline group,the hippocampal microglia in Meth group were significantly activated,and the activation of microglia was significantly inhibited after treatment with gastrodin of 10mg/kg,30mg/kg and 100mg/kg,and the inhibitory effect of gastrodin of 30mg/kg and 100mg/kg groups was more obvious.5.Effects of different doses of gastrodin on the expression of IL-1β and TNF-α in hippocampus of Meth-dependent CPP rats.Compared with Meth group,the protein expressions of IL-1β and TNF-α decreased in a dose-dependent manner after treatment with gastrodin of 10mg/kg,30mg/kg and 100mg/kg(P<0.01 or P<0.05),and the protein expressions of IL-1β and TNF-αshowed a dose-effect relationship with gastrodin administration.6.Effects of different doses of gastrodin on the expressions of key factors in TLR4/MyD88/NF-κB signal pathway in hippocampus of Meth-dependent CPP rats.Compared with Meth group,after treatment with gastrodin of 10mg/kg,30mg/kg and 100 mg/kg,gastrodin decreased the protein and mRNA expression of TLR4,MyD88,TRAF6,and NF-κBp65 in a dose-dependent manner(P<0.001,P<0.01 or P<0.05),increased the protein and mRNA expressions of IκB-α in a dose-dependent manner(P<0.01),decreased the protein expressions of NF-κBp65 in a dose-dependent manner(P<0.01 or P<0.05),decreased the protein expression of p-iκB-α(P<0.01).The expression of TLR4,MyD88,TRAF6,NF-κBp65,IκB-α,and NF-κBp65 showed a dose-effect with gastrodin,but the protein expression of p-iκB-α and the dose of gastrodin was no significant dose-effect relationship.Conclusion1.TLR4/MyD88/NF-κB signal pathway is involved in the formation of CPP in methamphetamine dependent rats.2.Methamphetamine induced neuroinflammation in hippocampus of methamphetamine-dependent CPP rat via TLR4/MyD88/NF-κB signal pathway.3.Gastrodin improved hippocampus neuroinflammation in methamphetamine-dependent CPP rat in a dose-dependent manner.4.The protective effect of gastrodin on neuroinflammation of hippocampus in methamphetamine dependent CPP rats is closely related to TLR4/MyD88/NF-κB signal pathway.