Anti-inflammatory Property Of Bicyclol In Rat Ischemic Stroke

Objective: Ischemic brain injury results from a sequence ofpathophysiological events, including inflammation, oxidative stress,excitotoxicity and apoptosis. Increasing evidences demonstrate thatinflammation is a critical determinant of outcome and might be the secondaryinjury mechanism following cerebral ischemia. Toll-like receptors(TLRs)-induced nuclear factor-kappa B (NF-κB) activation pathway has beenrecognized as a key contributor to the pro-inflammatory response. TLRs are amajor family of transmembrane pattern-recognition receptors (PRRs) that cansense the invasion of pathogenic microorganisms and tissue injury, a functionthought to be limited to immune cells. Recent studies revealed that TLR2,TLR4, and TLR9are induced in post-ischemic brain tissue. We have provedthat TLR2and TLR4, myeloid differentiation primary-response protein88(MyD88), and NF-κB activation as mechanisms linking to the pathogenesis ofcerebral ischemia. TLR9as an inflammatory mediator also take part in theprocess of ischemic damage. Tumor necrosis factor receptor associated factor6(TRAF6) is a member of the TRAFs family, which are required forMyD88-dependent TLRs activation. TRAF6interacts with the C-terminus ofIL-1R-associated kinase (IRAK)-1leading to the activation of downstreamsignaling pathways like the MAP kinase cascades and the NF-κB inducingpathway. The activation of NF-κB induces the up-regulation of leukocyteadhesion molecules and the production of pro-inflammatory cytokines, thereby,promoting inflammation.Bicyclol (4,4-dimethoxy-5,6,5,6-bis (dimethylene-dioxy)-2-hydroxymethyl-2-methoxy carbonyl biphenyl) derives from it’s precursorChinese herb Fructus Schizandrae. It has been reported that bicyclol hasprotective effect against experimental liver injury induced by various chemicaltoxins. The hepatoprotection of bicyclol involves the clearance of reactive oxygen species, regulation of cytokine secretion, and inhibition of apoptosisinduced by immunological injury. Recently, it was reported that bicyclolprotected the liver against ischemia/reperfusion (I/R) injury, by inhibiting lipidperoxidation and down-regulating intercellular adhesion molecular1(ICAM-1), NF-κB and TLR4. However, whether modulation of localinflammation in ischemic brain plays a role in the neuroprotection provided bybicyclol is not clear.The purpose of this study was to evaluate the potential of bicyclol toattenuate cerebral ischemic injury and the possible mechanisms in rats.Methods: Male Sprague-Dawley rats were randomly assigned to five groups:permanent middle cerebral artery occlusion (pMCAO), Vehicle (pMCAO+0.5%sodium carboxymethylcellulose), By-L (Vehicle+bicyclol50mg/kg),By-H (Vehicle+bicyclol100mg/kg) and Sham operated group. Bicyclol wasadministered intragastrically once a day for3days, after1h of bicyclolpretreatment on the third day; rat brain ischemia was induced by pMCAO.Neurological deficit, infarct volume, and brain edema were measured at24hafter stroke. Immunohistochemistry, Western blot and real-time were used todetect the expression of TLR4, TLR9, TRAF6, NF-κB and MMP-9,Claudin-5.Results:1Neurological deficit was examined and scored on a6-point scale at24hpMCAO and Mann-Whitney U test analysis was conducted (Fig.2). Comparedwith pMCAO and Vehicle group, the neurologic deficit scores in By-H groupwere significantly reduced (P <0.05). By contrast, there was no significanteffect in By-L group compared with pMCAO and Vehicle groups (P>0.05).2Wet-dry method was used to measure brain water content. Brain watercontent at24h after ischemia was shown in Fig.3. Bicyclol could reduce thebrain water content of ipsilateral hemispheres. In the sham-operated group, thebrain water content was78.89%±0.79%. Compared with pMCAO andVehicle group, By-H group significantly reduced the brain water content(By-H vs. pMCAO and Vehicle:83.38%±0.70%vs.85.73%±1.17%and 85.90%±1.01%, P <0.05). But no significant decrease was observed in By-Lgroup.3Infarct volume was measured by TTC at24h after ischemia. No infarctionwas observed in the sham operated group, while extensive lesion wasdeveloped in both striatum and lateral cortex in pMCAO and Vehicle group(Fig.4). In By-H group, infarct volume was significantly reduced comparedwith pMCAO and Vehicle group.(By-H vs. pMCAO and Vehicle:31.89%±6.16%vs.45.16%±3.94%and46.10%±4.36%, P <0.05Fig.5). However,no significantly difference was found between pMCAO or Vehicle group andBy-L group (P>0.05).4The localization of TLR4, TLR9, TRAF6and NF-κB protein was identifiedby immunohistochemistry after stroke. In By-H group, the number of positivecells of TLR4, TLR9, TRAF6and NF-κB was significantly decreasedcompared with pMCAO or Vehicle group. Moreover, positive nuclei of NF-κBwere also reduced and lots of cells labeled by NF-κB were stained only incytoplasm. However, there were no significant differences about the positivecells between pMCAO or Vehicle group and By-L group (Fig.6).We further analyzed the protein and mRNA level of total TLR4, TLR9,TRAF6, nuclear NF-κB and MMP-9, Claudin-5with Western blot andRT-qPCR. Compared with pMCAO and Vehicle group, By-H groupsignificantly decreased the expression of TLR4, TLR9, TRAF6, nuclearNF-κB p65and MMP-9at protein level and mRNA levels (P <0.05Fig.7-11,13-14). However, there were no significant differences in the expression ofTLR4, TLR9, TRAF6, nuclear NF-κB p65and MMP-9between pMCAO orVehicle group and By-L group (P>0.05). Compared with Sham group,pMCAO induced greatly reduction of claudin-5(P <0.05). After pretreatmentwith high dose of bicyclol, the expression of Claudin-5was significantlyup-regulated at both protein and mRNA levels (P <0.05Fig.12,14), while theup-regulation of Claudin-5was not observed in low dose group (P>0.05).Conclusions: After systemic administration of bicyclol, the neurologic deficit scores, brain edema and infarct volume were reduced after ischemia,moreover, the expression of TLR4, TLR9, TRAF6, NF-κB and MMP-9wassignificantly decreased after stroke, indicating the potent anti-inflammatoryactivity of bicyclol in ischemic brain tissue. Those neuroprotective effects maybe through down-regulation of TLR4, TLR9, TRAF6, NF-κB and MMP-9,up-regulation of Claudin-5. TLRs/TRAF6/NF-κB pathway of may be one ofthe bicyclol’s effective therapeutic targets for sencondary injury in the acutephase of cerebral ischemia.