Protection Of Inositol Hexaphosphate In Cerebral Ischemia/Reperfusion Mice: Upregulated P-Akt And Claudin-5, Downregulated NF-κB And MMP-9Expression

Objective: Acute ischemic stroke is a mainly cause of death and the mostfrequent cause of permanent disability in adult worldwide. At present,although progress has been made in understanding the molecular pathwaysthat lead to ischemic cell death, therapeutic choices for acute stroke are stilllimited.Inflammatory and apoptosis damage play an important role in cerebralischemic pathogenesis and may represent a target for treatment. Experimentalevidence suggests that neurons at the border of the ischemic territory cansurvive for many hours, even days after an ischemic insult. The delayed tissueresponse to brain ischemia encompasses processes such as inflammation andapoptosis. Apoptosis, a special form of cell death, occurs after ischemia isdependent on the balance between apoptotic and survival signals. Evidencehas been presented that apoptosis appears in the peripheral penumbra ofischemia and phosphatidylinositol3kinase/protein kinase B (PI3K/Akt)pathway mediates neuronal survival after cerebral ischemia and reperfusion.Phosphorylation of Akt promotes cell survival against cerebral ischemic insultby phosphorylation and subsequent inactivation of many proapoptotic proteins,such as glycogen synthase kinse3β (GSK3β). GSK3β regulated theinflammatory response by differentially affecting the nuclear amounts oftranscription factors NF-κB subunit p65. Nuclear factor-kappa B (NF-κB) is aubiquitously expressed transcription factor that regulates expression of genesinvolved in inflammation, cell survival and apoptosis. Ample evidenceindicates that NF-κB is activated in cerebral ischemia and reperfusion (I/R),especially in neurons. Also many data suggest that inhibition of NF-κBreduces infarction volume and develops less ischemic damage in permanent ischemia especially NF-κB regulates the expression of both proinflammatorygenes, such as matrix metalloproteinase-9(MMP-9). Excessive MMP activityis deleterious-direct injection of MMP-9into brain causes cell death andinflammation.Inositol hexaphosphate (IP6), a phytochemical found in food grains hasgained attention for its multiple beneficial biological effects through itsanti-inflammatory, anti-oxidant and anti-tumor propertiestebie, particularly, itsprotective effects for myocardial reperfusion. Currently, little is known aboutthe effect of IP6in cerebral ischemia/reperfusion (I/R), therefore weinvestigated the potential neuroprotective effects of IP6in cerebral I/R and theunderlying mechanisms.Methods: Male CD1mice were subjected to transient middle cerebralartery occlusion (tMCAO) and randomly assigned into four groups: Sham,tMCAO (0.9%saline), IP6–L (tMCAO+IP675mg/kg) and IP6-H (tMCAO+IP6125mg/kg) groups. IP6was administered intraperitoneal injectionimmediately after tMCAO for IP6–L and IP6-H groups, while Sham andtMCAO groups were administered the equal volume normal saline. Theneurological deficit, brain water content and infarct size were detected at24hafter ischemia/reperfusion. The expression of phospho-Akt (p-Akt), nuclearfactor-kappB (NF-κB), matrix metalloproteinase9(MMP-9) and claudin-5inischemic cerebral cortex were detected by immunohistochemistry, westernblotting and RT-qPCR. Cell death characteristic for tMCAO model weredetected by Nissl histology.Results:1The neurological deficit: Neurological deficit was examined and scoredon a5-point scale at24h after tMCAO,24mice were evaluated in each group.Compared with Sham group, the neurological deficit scores were significantlyhigher in tMCAO group. Compared with tMCAO control, IP6significantlyattenuated neurological deficit in IP6-L and IP6-H (IP6-L vs. tMCAO:3.16± vs. IP6-H:3.16±0.70vs.2.67±0.87P<0.05).2The brain infarction volume: No infarction was observed in thesham-operated group. In tMCAO group, an extensive lesion was developed inlateral cortex. The infarct volume was significantly reduced from62.45%±9.41%in tMCAO group to47.20%±6.16%in the IP6-L group (P <0.01) and34.59%±7.37%in the IP6-H group (P <0.01), and IP6-H group weresignificantly lower than IP6-L group(IP6-L vs. IP6-H:47.20%±6.16%vs.34.59%±7.37%P<0.05).3The brain edema: Ipsilateral brain water content of Sham group was79.80%±0.98%. Compared with tMCAO group, IP6-L and IP6-Hsignificantly decreased the brain water content (tMCAO vs. IP6-L:85.12%±1.67%vs.82.73%±1.62%, P <0.05; tMCAO vs. IP6-H:85.12%±1.67%vs.81.13%±0.72%, P <0.05), and IP6-H group were significantly lower thanIP6-L group (IP6-L vs. IP6-H:82.73%±1.62%vs.81.13%±0.72%, P <0.05).4IP6upregulated the expression of p-Akt and claudin-5anddownregulated the expression of NF-κB (p65) and MMP-9: Outcome ofimmunohistochemistry showed that the number of positive cells of p-Akt,dramatically increased in ischemic cortex of IP6-L and IP6-H groups. IP6significantly reduced the positive cells of NF-κB and MMP-9after tMCAO inIP6-L and IP6-H groups. In agreement with results of immunohistochemistry,the expression of p-Akt and claudin-5was upregulated and the expression ofNF-κB (p65) and MMP-9was suppressed at protein level after systemicadministration of IP6in ischemia brain. In agreement with results of westernblot, simultaneously, RT-qPCR analyses also showed a significant decrease ofNF-κB and MMP-9in IP6-L group and IP6-H group.5IP6reduced the cells apoptosis: Nissl histology revealed delayed celldeath characteristic for tMCAO model. The sham group did not exhibit deadcells. Cell loss and the presence of injured neurons with twisted axonalprocesses were noted in the tMCAO group. In the IP6-L group and IP6-Hgroup relatively less dead cells were observed compared with tMCAO group. 6IP6protected the integrity of BBB: The integrity of brain blood barrier(BBB) was assessed by claudin-5and MMP-9which was primarily present inbrain tight junctions of endothelia. Compared with tMCAO group, claudin-5’sexpression was increased, and MMP-9’s expression was reduced in both IP6-Land IP6-H groups, by western blotting and RT-qPCR at24h.Conclusions: IP6attenuated neurological deficit, infarction volume andbrain edema protected the brain against cerebral ischemic and this effect maybe through upregulating the expression of p-Akt and claudin-5,downregulating NF-κB and MMP-9and protected the integrity of BBB.