Role Of TLR4-Peli1 Signaling Pathway In Methamphetamine-induced Microglial Inflammatory Response

Background Methamphetamine(Meth),commonly known as methamphetamine,is one of the most common mental stimulants.The nervous system is one of the major target systems of Meth.In recent years,molecular biology techniques and in vivo imaging techniques have been used.Neuroinflammation is mainly a physiological reaction caused by excessive activation of microglia in the brain.Peli1 protein has the role of E3 ubiquitinating ligase and has specificity in the microglia inflammatory reaction.TLR4 is the main receptor on the surface of microglia and can activate MyD88 and TRIF pathways,and activate the NF-κB pathway to promote the expression of inflammatory factors.It has been reported in the literature that Peli1 activates the TRIF-dependent TLR signaling pathway and promotes the release of inflammatory factors.Our previous experiments showed that Meth can activate microglia,increase the expression of TLR4 and Peli1 protein,induced the activation of MAPKs and NF-κB pathway,and caused the release of inflammatory factors,resulting in an inflammatory response.Therefore,we speculate that TLR4-Peli1 may be as a key signal axis,it plays an important role in Meth-induced neuroinflammatory reactions.Objective The role of TLR4-Peli1 signaling pathway in Meth induced microglial inflammatory responses.Methods A mouse model of acute Meth exposure was established and randomly assigned to three dose groups of 17 in each group,control(saline),low-dose Meth(10 mg/kg)and high-dose Meth(30 mg/kg).)Intraperitoneal injections were administered four times a day for a period of time of eight o’clock,ten o’clock,twelve o’clock and two o’clock in the afternoon.Four high-dose Meth group(30 mg/kg)mouse died after administration.Using western blot to detected the changes of TLR4,Peli1,MyD88,and TRIF proteins and the activation of MAPKs and NF-κB pathways.Using lentivirus transfection technology to down-expressed Peli1 on BV2 cells,and detected the changes of MAPKs and NF-κB pathways,and the secretion levels of cytokines IL-6,IL-12P70 and TNF-α.The effect of TAK242 on the viability of BV2 cells was measured using the CCK-8 kit,and the optimal dose of TAK242 was screened without affecting cell viability.Through the inhibition and knockdown of TLR4,observed the changes of Peli1 protein and downstream phosphorylation of MAPKs,NF-κB pathway and the secretion levels of cytokines IL-6,TNF-α and IL-1β.Gene knockdown of TRIF and MyD88 was performed on BV2 cells by si RNA interference technique,and the changes of Peli1 protein were observed by RT-PCR and western blot.Results 1.Effects of methamphetamine on TLR4,Peli1 and signaling pathways in mouse hippocampus TLR4,MyD88,TRIF,and Peli1 showed significant up-regulation of protein expression in low-dose Meth(10 mg/kg)and high-dose Meth(30 mg/kg)groups.Compared with the control group,low dose group meth(10 mg/kg)and high dose group(30 mg/kg)can promote different degrees of activation of MAPK pathway,manifested as p-ERK,p-JNK and p-p38 protein expression increased.The expression of NF-κB phosphorylation protein was significantly increased.2.The role of Peli1 in methamphetamine-induced microglial inflammatory response The Peli1 protein expression level in the Peli1 knockdown group was significantly lower than that in the empty vector group.Meth(300 μM),compared with the empty vector group,the phosphorylation level of NF-κB was significantly decreased in the Meth group after Peli1 knockdown.After low expression of Peli1 and Meth(300 μM),the phosphorylation level of ERK in the empty vector transfection group was significantly lower than that in the Pel1 transfection group(P<0.01),Phosphorylation of JNK in the infected group also decreased(p<0.05).In the Meth group knocked down by Peli1,the levels of IL-6,IL-12P70,and TNF-αwere decreased in the Meth group compared with the empty vector group.3.Effect of TLR4 inhibitor TAK242 on Meth-induced microglial TLR4 protein and its downstream signaling pathway20 μM of TAK242 began to have a damaging effect on BV2 cells,and 30 μM of TAK242 acting dose significantly reduced cell viability.Different concentrations of TAK242 had a significant inhibitory effect on TLR4 expression,especially at a concentration of 15 μM,TLR4 had the most obvious inhibitory effect,and downstream MyD88 and TRIF also showed different degrees of reduction.After TLR4 inhibition,MAPKs pathway(ERK,p38)Phosphorylation levels of JNK and JNK decreased to varying degrees,and NF-κB phosphorylation levels also decreased significantly.After TAK242(15 μM)was screened,the expression of TLR4 protein decreased significantly,MyD88,TRIF,and Peli1 also showed a decreasing trend,and the phosphorylation levels of MAPKs(ERK,p38,JNK MAPK)decreased to varying degrees.NF-κB Phosphorylation levels decreased.After pre-addition of TAK242(15μM),the expression of IL-6,TNF-α,IL-1β,and other inflammatory cytokines was significantly reduced by Meth.4.Effect of siRNA interfering with TLR4 on Meth-induced microglial inflammatory response and its downstream signal proteins The protein level in the TLR4 interference group was significantly lower than that in the empty vector group,indicating that the interference effect was better.Afterwards,we observed the expression of the MyD88 and TRIF proteins on the downstream MyD88-dependent and non-independent pathways in response to TLR4 interference.The results showed that compared with the Meth-treated group alone,the TLR4 interfered with Meth after MyD88 and TRIF.And the expression of downstream Peli1 declined.Furthermore,after administration,compared with the empty vector group,the phosphorylation levels of the MAPKs pathway(ERK,p38MAPK)and NF-κB in the TLR4 interference group were reduced to different degrees,and the results were basically consistent with those of the TLR4 inhibitor.5.Changes of Peli1 in microglia-induced microglial cells induced by MyD88 dependent and non-dependent channels After si RNA interference by MyD88,the m RNA and protein expression of MyD88 in the interfering group was significantly lower than that in the empty vector group,indicating that the MyD88 interference was better.After si RNA interference with MyD88,the expression of Peli1 m RNA and protein did not change significantly.Consistent with the results of m RNA expression levels.By interfering TRIF with si RNA,followed by Meth(300 μM)for 24 h,TRIF m RNA levels and protein levels were decreased after TRIF interference group compared with empty vector,and the m RNA and protein levels of Peli1 in the TRIF interference group accompanied decreased.Conclusions1.The expression of TLR4,TRIF,MyD88,and Peli1 proteins increased in the hippocampus of mice acutely treated with Meth,and the MAPKs and NF-κB pathways were activated.The expression of ERK,JNK,P38,and NF-κB phosphorylation was increased,suggesting that Meth mediated neuroinflammation.2.Peli1 is a receptor that is mainly expressed on the surface of microglial cells of Peli family members.When it is knocked down,the phosphorylation levels of downstream MAPKs and NF-κB pathways are decreased,and IL-6,TNF-α,and IL-12P70 are involved in cell inflammation.It is suggested that in Meth-induced microglial cells,Peli1 may serve as a key point to mediate inflammatory effects.3.As one of the major Toll-like family receptors on the surface of microglia,TLR4 regulates the secretion of downstream inflammatory pathways and inflammatory cytokines during Meth-induced microglial inflammatory responses.After TLR4 inhibition,the level of Peli1 decreased and the phosphorylation levels of MAPKs and NF-κB pathways decreased.This suggests that TLR4 may regulate the inflammatory response of Meth-induced microglial cells upstream of Peli1.4.Interfering with MyD88 and TRIF,respectively,and found that Meth-induced microglial inflammatory response,TLR4 regulation of Peli1 may be achieved through the MyD88-independent TRIF pathway.