The Neuroprotective Effects And Mechanisms Of (+)-borneol In A Rat Model Of Pilocarpine-induced Epileptogenesis

Objective:To explore the neuroprotective and potential mechanisms of(+)-borneol in a rat model of pilocarpine-induced epileptogenesis.Methods:1.To explore the effects of different concentrations of(+)-borneol on the expression of inflammatory factors in the hippocampus of rats with pilocarpine-induced epileptogenesis and select the appropriate concentration of(+)-borneol for the next study.30 healthy male SD rats(weighing 250-300g)were randomly divided into 5 groups(n=6):(1)control group;(2)status epilepticus(SE)group;(3)SE + low dose(+)-borneol(3mg/kg)group;(4)SE + medium dose(+)-borneol(6mg/kg)group;(5)SE + high dose(+)-borneol(12mg/kg)group.The SE model was induced with pilocarpine,and the SE+(+)-borneol group was injected intraperitoneally with different concentrations of(+)-borneol solution at the same time every day starting 60 minutes after SE.The same volume of vehicle was given to the control and SE groups.After 7 days of administration,the brains were severed and removed.The expression of pro-inflammatory factors TNF-α,IL-1β and COX-2 in hippocampal was detected using ELISA and Western Blot.The appropriate concentration of(+)-borneol was selected for the next step of the study.2.To explore the effects and possible mechanisms of(+)-borneol on glial cell activation,neuronal damage and apoptosis in the hippocampus of rats with pilocarpine-induced epileptogenesis.54 healthy male SD rats(weighing 250-300 g)were randomly divided into 3 groups(n=18):(1)control group;(2)SE group;(3)SE+(+)-borneol group.The groups were divided into 3 subgroups(1d,3d and 7d,n=6 for each subgroup)according to the different time points of the assay.The SE model was induced with pilocarpine,and the SE+(+)-borneol group was injected intraperitoneally with(+)-borneol solution at the same time every day starting 60 minutes after SE.The same volume of vehicle was given to the control and SE groups.The brains were severed at 1d,3d and 7d after administration.The astrocyte marker GFAP,microglia marker Iba-1 and neuronal marker NEUN were detected by Western Blot and immunohistochemistry,and the apoptosis-related proteins BAX and Bcl-2,NF-κB signaling pathway-related proteins(IκB-α,P65,P-P65)were detected by Western Blot.Results:1.Expression of inflammatory factors: Compared with the control group,the expression levels of TNF-α,IL-1β and COX-2 in the hippocampus of rats in the SE group were significantly increased(P < 0.001).The expression levels of TNF-α,IL-1βand COX-2 were significantly reduced in the low,middle and high doses of(+)-borneol groups compared with the SE group.(+)-borneol had a stronger effect in the high dose group compared to the rest of the dose groups(P<0.001).2.(1)Glial cell activation and neuronal damage: Both Western Blot and immunohistochemistry results showed that there was no statistical difference in GFAP,Iba-1 and NEUN expression between different time points(1d,3d and 7d)in the control group(P>0.05).Compared to the control group,NEUN expression was reduced and GFAP and Iba-1 expression was increased in the SE group(P<0.001)in a time-dependent pattern.Compared with the SE group,the(+)-borneol group significantly increased the expression of NEUN and decreased the expression of GFAP and Iba-1(P <0.05).(2)Apoptosis-associated protein expression: Western Blot results showed that there was no statistical difference in BAX/Bcl-2 levels between different time points(1d,3d,7d)in the control group(P > 0.05).BAX/Bcl-2 levels were significantly higher in the SE group compared to the control group,with an approximately 10-fold increase at day 7(P< 0.001).BAX/Bcl-2 levels were significantly decreased in the(+)-borneol group compared to the SE group(P < 0.001).(3)Expression of NF-κB signaling pathway: Western Blot results showed that there was no statistical difference in the expression of IκBα and P-P65/P65 between different time points(1d,3d,7d)in the control group(P > 0.05).Compared to the control group,IκBα expression was reduced and P-P65/P65 levels were increased in the SE group(P <0.001).Compared with the SE group,there was no statistical difference in IκBαexpression between the(+)-borneol group and the SE group at 1 d of administration(P >0.05).The(+)-borneol group significantly increased the expression of IκBα at 3 d and 7 d of administration(P < 0.001).The P-P65/P65 levels were reduced in the(+)-borneol group compared to the SE group(P < 0.05).Conclusion:(+)-Borneol can inhibit inflammatory factor expression,glial cell activation,neuronal damage and apoptosis in the hippocampus during epileptogenesis after pilocarpine-induced SE,and can inhibit the activation of NF-κB pathway,thus exerting neuroprotective effects.Therefore,(+)-Borneol may have an inhibitory effect in epileptogenesis and is a potential disease-modifying therapeutic agent for epilepsy.