| Objective: Cerebral ischemia is the most common type ofcerebrovascular disease, which is the first leading cause of death and the mostfrequent cause of permanent disability in adults. The most effective way tocontain cerebral ischemic injury is reperfusion, however, reperfusion itselfmay result in tissue injury, for which inflammatory damage is one of maincausative factors. NALP3inflammasome is a multiprotein complex. It consistsof NACHT domain-, leucine-rich repeat-, and pyrin domain (PYD)-containingprotein3(NALP3), apoptosis-associated speck-like protein containing acaspase recruitment domain (ASC) and caspase-1, whose function is to switchon the inflammatory process.Chrysophanol (CHR) is an extract from plants of Rheum genus and itpossesses many pharmacological effects including its anti-inflammationactivity, for which the underlying mechanisms remain to be elucidated. In thisstudy, the effects of chrysophanol in cerebral ischemia/reperfusion (I/R) andthe potential mechanisms were investigated. Male CD1mice were subject totransient middle cerebral artery occlusion (tMCAO). The NALP3inflammasome activation status and its dynamic expression during the naturalinflammatory response induced by tMCAO were first profiled. Theneuroprotective effects of CHR were then assessed and the potentialmechanisms mediating the observed neuroprotection were then explored.Methods: Male CD1mice (25-30g) were subject to tMCAO, asdescribed by Longa previously.Experiment1was used to determine whether the NALP3inflammasomewas activated in inflammatory response induced by tMCAO, and its dynamicexpression was evaluated.63mice were divided into7groups randomlywhich included Sham group, and groups of six time points (3h,6h,12h,24h, 48h,72h) after tMCAO, n=9for each group.Experiment2was applied to detect the neuroprotection of CHR and itspotential mechanism.162mice were divided into6groups randomly. Shamgroup: animals received Sham operation and equal volume of NaCl0.9%;tMCAO group: animals received tMCAO and equal volume of NaCl0.9%;Vehicle group: animals received tMCAO and equal volume of DMSO andTween-80; and CHR groups: animals received tMCAO and treated with highdose of CHR10mg/kg (CHR-H), middle dose of CHR1mg/kg (CHR-M) andlow dose of CHR0.1mg/kg (CHR-L).Drug or solvent was injected intraperitoneally30minutes prior totMCAO operation. Neurological deficit was examined and scored at24h aftertMCAO. And then mice were reanesthetized for sacrificing. Wet-dry methodwas used index to measure brain edema; Infarct volume of each group wasmeasured by TTC; BBB leakage was measured using Evans blue extravasation.Confocal microscopy, western blotting, immunohistochemistry and qRT-PCRtechniques were utilized to analyze the expression of NALP3inflammasomeand IL-1β.Results:1NALP3, ASC, Caspase-1and IL-1β were in neuronsConfocal microscopy was utilized to detect the presence and the cellulardistribution of NALP3inflammasome components as well as IL-1β in theischemic penumbra zone of cerebral cortex. The neuronal karyopyknosis andchromatin margination could be observed and there was a small amount ofneutrophils in marginal zone of infarction. In the normal brain (Sham),caspase-1and NALP3were mainly localized in the nucleus of neurons,whereas IL-1β was mainly present in the cytoplasm. ASC was present in bothof nucleus and cytoplasm. However, after tMCAO, NALP3and caspase-1were redistributed predominantly to the cytoplasm, while ASC and IL-1βlocation remained unchanged.(Fig.3)2NALP3, ASC, Caspase-1and IL-1β were up-regulated during cerebralI/R Immunohistochemistry, qRT-PCR and western blotting were used toexamine expression of NALP3, ASC, caspase-1and IL-1β in brain tissue aftertMCAO. The results showed that NALP3and active caspase-1were bothup-regulated as compared with Sham group (P <0.05), beginning at12h andpeaking at24h (Fig4a,4b,4d,4e,4f). In contrast, active IL-1β was alsoshown to be up-regulated but with an earlier onset at6h post tMCAO. ASCremained unchanged until72h after tMCAO (Fig4c,4d,4e,4f). Therefore,we took the time point of24h after tMCAO to observe the effects of CHR, soas to demonstrate the protective effects of CHR on acute cerebral I/R.3CHR reduced neurological deficitsNeurological deficit was examined and scored on a6-point scale at24hafter tMCAO. As depicted in Fig5a, mice in Sham group showed aneurological score as zero while the mice from the tMCAO and Vehiclegroups showed expected higher neurological deficit scores after the surgery ascompared with Sham group (P <0.05). No significant difference was observedbetween the animals from tMCAO and Vehicle groups (P <0.05). Remarkably,mice from the CHR-H and CHR-M groups showed significantly improvedneurological function scores as compared with tMCAO and Vehicle groupsafter tMCAO (P <0.05). By contrast, there was no significant effect in CHR-Lgroup compared with tMCAO and Vehicle groups (P>0.05).4CHR reduced the infarct volumeInfarct volume of each group was measured by TTC at24h after tMCAO(Fig.5b). Significant protective effect from CHR was observed as the infarctvolumes from animals in the CHR-H and CHR-M groups, but not the CHR-Lgroup (P>0.05), are significantly different from the tMCAO and Vehiclegroups (Fig.5c). There was a significant reduction in infarct volume in CHR-H(29.80%±1.15%vs.49.87%±1.99%and51.31%±1.57%, P <0.05) andCHR-M (41.86%±0.98%vs.49.87%±1.99%and51.31%±1.57%, P <0.05)groups as compared with tMCAO and Vehicle groups.5CHR reduced the brain edemaIt was demonstrated that no change in brain edema in the Sham group, but a significant increase in brain water content in the ischemic hemispheres inthe tMCAO and Vehicle groups as compared with those from the Sham group(P <0.05). Following CHR treatment, as compared with tMCAO and Vehiclegroups, there were significant reductions in brain water content in the CHR-Hgroup (80.60%±0.58%vs.83.75%±0.48%and83.72%±0.35%, P <0.05)and the CHR-M group (82.15%±0.68%vs.83.75%±0.48%and83.72%±0.35%, P <0.05). Again, no significant difference was observed between theCHR-L group and the tMCAO and Vehicle groups (83.88%±0.31%vs.83.75%±0.48%and83.72%±0.35%, P>0.05)(Fig.5d).6The amelioration of BBB permeabilityAs expected, an extensive BBB disruption was found in animals from thetMCAO and Vehicle groups (P <0.05vs. Sham group). The protective effectsof CHR with different doses were also observed by examination of Evans bluecontent at24h and, as compared with the tMCAO and Vehicle groups,significantly lower Evans blue extravasation was observed in the CHR-H andCHR-M groups (P <0.05vs. tMCAO and Vehicle). In agreement withneurological deficit, infarct volume and brain edema measurement, the CHR-Lgroup didn’t show significant difference in comparison with the tMCAO andVehicle groups (P>0.05)(Fig.5e,5f).7CHR suppressed the expression of NALP3, caspase-1and IL-1βThe immunohistochemical staining of NALP3, ASC, caspase-1and IL-1βof each group at24h after operation was shown in Fig6. Few cells wereexpressing NALP3, caspase-1or IL-1β in the cortex in Sham group indicatinga low baseline of NALP3, caspase-1and IL-1β expressions in non-ischemiccortex. In contrast, tissues from the tMCAO group showed an augmentedexpression of all three examined proteins. The number of cells expressingNALP3, caspase-1or IL-1β in the ischemic cortex of tMCAO mice were allsignificantly increased as compared with Sham group (P <0.05). Similarresults were also obtained from the Vehicle group and no significant differencewas observed between the tMCAO and Vehicle groups (P>0.05)(Fig7e). InCHR-H and CHR-M groups, the number of cells positive for NALP3, caspase-1or IL-1β were all significantly lower than the tMCAO and Vehiclegroups (P <0.05)(Fig.7e). Then we further analyzed the protein expression ofNALP3, ASC (Fig.7a), caspase-1(Fig.7b) and IL-1β (Fig.7c) with westernblotting. It was found that the expressions of NALP3, caspase-1and IL-1β atprotein level were up-regulated in tMCAO and Vehicle groups compared withSham group (P <0.05). High dose and middle dose CHR significantlydecreased the NALP3, caspase-1and IL-1β protein expression compared withtMCAO and Vehicle groups (P <0.05)(Fig.7a,7b,7c). In agreement with theresults of immunohistochemistry and western blotting, the mRNA expressionof NALP3, caspase-1and IL-1β were up-regulated in tMCAO and Vehiclegroups compared with Sham group (P <0.05). The over-expression of thosefactors were significantly decreased in CHR-H and CHR-M groups comparedwith tMCAO and Vehicle groups (P <0.05)(Fig.7f).Conclusions: NALP3inflammasome activation was accompanied withthe damage of brain tissue in cerebral I/R. CHR could inhibit the activation ofNALP3inflammasome and protect the cerebral ischemic stroke. |
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