Crocetin Inhibits The Activation Of Kainic Acid-induced Microglia Via TLR4/NF-κB/NLRP3 Pathway

Objective: Microglia activation is one of the important mechanisms for the progressive development of epilepsy.Inhibition of microglial overactivation is the key link in the prevention and treatment of the progressive development of epilepsy.The purpose of this paper is to observe the inhibitory effect of crocetin(CRO)on kainic acid(KA)-induced microglial activation,clarify the specific mechanism of crocetin inhibiting this activation through TLR4/NF-κB/NLRP3 signaling pathway,and preliminarily verify the protective effect of crocetin in epilepsy model rats at the animal level,and provide a new strategy for the progressive development of epilepsy.Methods:(1)In vitro experiment,BV-2 microglia were divided into CON group,KA group,CRO+KA group,The TLR4 inhibitor-pretreated group(TAK242+KA group)and crocetin group(CRO group).The proliferation of BV-2 cells was detected by CCK8.The ultrastructure of BV-2 cells was observed by electron microscopy.ATP level of BV-2 cells was detected by chemiluminescence.BV-2 cells were counted by trypan blue staining method.Detection of reactive oxygen species(ROS)in BV-2 cells using DCFH-DA fluorescent probe method and detection of mitochondrial ROS(mt ROS)levels in BV-2 cells using the Mito SOX fluorescent probe method.TLR4,p65,NLRP3,ASC,Caspase-1,IL-1β,TNF-α,IL-6,IL-18,i NOS m RNA expression in BV-2 cells were detected by qRT-PCR.Iba1,CD68,TLR4,p-p65,NLRP3,ASC,Caspase-1p20 protein expression in BV-2 cells were detected by Western blot.IL-1β,TNF-α,IL-6,IL-18,i NOS protein level in BV-2 cells were detected by ELISA.(2)In vivo experiment,Eighteen Wistar rats were divided into the control group(CON group),kainic acid group(KA group)and crocetin-pretreated group(CRO+KA group).Rat behavioral scores were performed according to the Racine grading criteria.Immunofluorescence staining to detect the number of activated microglia and neurons in the rat hippocampus.Results:(1)Electron microscopy showed that BV-2 cells in KA group had increased volume,enlarged nucleus,mitochondrial swelling and increased numbers compared with CON group.BV-2 cell in CRO+KA group had decreased volume,decreased nucleus,reduced mitochondrial swelling with decreased numbers compared with KA group.ATP assay results showed that ATP levels were significantly higher in KA group compared to the CON group and significantly lower in CRO+KA group compared to KA group;Trypan blue count showed increased BV-2 cells in KA group,pretreatment with CRO significantly reduced the BV-2 cell number;Western blot results showed that CRO significantly inhibited KA-induced increase in the expression levels of Iba1 and CD68 proteins.(2)The results of ROS showed that the intracellular total ROS levels and mt ROS levels were obviously increased in KA group compared with CON group.The intracellular total ROS levels and mt ROS levels in CRO+KA group were obviously reduced compared with KA group.(3)The results of qRT-PCR showed that the expression levels of TLR4,p65,NLRP3,ASC and Caspase-1 m RNA raised obviously in KA group compared with CON group,while the expression levels of TLR4,p65,NLRP3,ASC and Caspase-1 m RNA were obviously lower in CRO+KA and TAK242+KA groups compared with KA group.Western blot results showed that CRO and TAK242 significantly inhibited KA-induced increase in TLR4,p-p65,NLRP3,ASC and Caspase-1p20 protein expression levels.(4)The results of the inflammatory factor assay indicated that m RNA expression levels and protein levels of IL-1β,TNF-α,IL-6,IL-18 and i NOS increased obviously in the KA group compared with CON group.The m RNA expression levels and protein levels of IL-1β,TNF-α(11)IL-6,IL-18,and i NOS decreased obviously in CRO+KA and TAK242+KA group compared with KA group.(5)Animal results showed that the time of grade I-V epileptic seizures in CRO + KA group was later than that in KA group,and the duration of seizures was shorter than that in KA group;The number of activated microglia of hippocampus in CRO+KA group was obviously lower than that in KA group,while was the number of neurons obviously higher than that in the KA group.Conclusion:(1)Crocetin inhibits kainic acid-induced microglial activation and proliferation(2)Crocetin may inhibit kainic acid-induced microglial activation and inflammatory cytokine production via TLR4/NF-κB/NLRP3 pathway.(3)Crocetin can inhibit microglial activation,reduce neuronal damage in kainic acid-induced epileptic rats and improve epileptic seizures.