Effects Of Naringenin On Focal Cerebral Ischemic Injury And Expression Of NOD2, RIP2, NF-κB, MMP-9and Claudin-5in Rats

Objective: Cerebrovascular disease is considered to be one of the threeleading causes of death in the world, especially ischemic cerebrovasculardisease, with high rate of morbidity, disability, mortality and recurrence,posing a serious threat to human life. Brain tissue injuries secondary toischemia, which include inflammation, oxidative stress, calcium overload,glutamate excitotoxicity, apoptosis and mitochondrial dysfunction, aggravatethe illness and inhibit the recovery of patients. Thereinto, the role ofinflammatory response in cerebrovascular disease is gaining increasingattention, how to reduce the post-infarcted inflammatory response becomes animportant therapy. In recent years, with further study on immune inflammation,the nucleotide oligomerization domain-like receptors2(NOD2)/receptor-interacting protein2(RIP2) induced nuclear factor kappa B(NF-κB) activation pathway is supposed to participate in the inflammatoryresponse secondary to cerebral ischemia, while down-regulating this pathwaycould reduce the inflammatory response after ischemia and offerneuroprotection. Ample pharmacological and clinical studies have shownnaringenin possesses a vanity of pharmacological effects, such asanti-inflammatory, anti-oxidative, anti-apoptotic, anti-viral, anti-tumor and soon. As naringenin is liposoluble, it exhibits high permeability across theblood-brain barrier, the neuroprotective effects of naringenin on the centralnervous system have been well documented in Parkinson’s disease andAlzheimer’s disease. However, there is still a paucity of data regarding theeffect of naringenin in the acute phase of ischemic stroke. The purpose of thisstudy is to evaluate the potential neuroprotective effect of naringenin andunderlying mechanisms after cerebral ischemia induced in male adultSprague-Dawly rats by permanent middle cerebral artery occlusion (pMCAO). Methods: Healthy, male Sprague-Dawley rats were subjected tomodified permanent MCAO as described by Longa previously.120rats wererandomly assigned into Sham operated group (Sham), pMCAO group(pMCAO), pMCAO with vehicle group (Vehicle), pMCAO with low-dose (50mg/kg per day) naringenin group (NG-L) and pMCAO with high-dose (100mg/kg per day) naringenin group (NG-H). At24h after ischemia, neurologicaldeficit was evaluated, brain water content was measured by wet-dry method,infarct size were analyzed with2,3,5-triphenyltetrazolium chloride (TTC)staining; immunohistochemistry, western blot and real-time reversetranscription-quantitative PCR (RT-qPCR) were used to detect the variation ofNOD2, RIP2, NF-κB, MMP-9and claudin-5in ischemic cerebral cortex.Results:1Rats in Sham group had a neurological score of zero at all time points.Rats in pMCAO group, Vehicle group, low dose group and high dose groupperformed a right palsy. Neurological deficit score in high dose group wassignificantly decreased compared with pMCAO and Vehicle groups (pMCAOgroup vs. NG-H group:3.38±0.16vs.2.67±0.20, P <0.05; Vehicle group vs.NG-H group:3.42±0.13vs.2.67±0.20, P <0.05). There was a decrease inthe neurological deficit score following low dose NG administration, but therewas no significant difference in low dose group compared with pMCAO andVehicle groups (pMCAO group vs. NG-L group:3.38±0.16±vs.3.04±0.13,P>0.05; Vehicle group vs. NG-H group:3.42±0.13vs.3.04±0.13, P>0.05).2The brain water content in high dose group was significantly decreasedcompared with that in pMCAO and Vehicle groups at24h (pMCAO group vs.NG-H group:85.46±0.40%vs.83.17±0.55%, P <0.05; Vehicle groupvs. NG-H group:85.15±0.32%vs.83.17±0.55%, P <0.05). There was adecrease in the brain water content following low dose NG administration, butthere was no significant effect in NG-L group compared with pMCAO andVehicle groups at24h (pMCAO group vs. NG-L group:85.46±0.40%vs.84.36±0.38%, P>0.05; Vehicle group vs. NG-H group:85.15±0.32%vs. 84.36±0.38%, P>0.05).3Infarct size was significantly decreased in NG-H group compared withthat in pMCAO group and Vehicle group at24h (pMCAO group vs. NG-Hgroup:44.35±2.23vs.34.44±1.65, P <0.05; Vehicle group vs. NG-H group:44.42±1.53vs.34.44±1.65, P <0.05). There was a decrease in the infarctsize following low dose NG administration, but no statistical significance inNG-L group compared with pMCAO and Vehicle groups at24h (pMCAOgroup vs. NG-L group:44.35±2.23vs.38.99±2.54, P>0.05; Vehicle groupvs. NG-H group:44.42±1.53vs.38.99±2.54, P>0.05).4High dose of naringenin significantly down-regulated the expression ofNOD2, RIP2, NF-κB and MMP-9, up-regulated the expression of claudin-5at24h after ischemia (P <0.05). Low dose of naringenin can alsodown-regulated the expression of NOD2, RIP2, NF-κ B and MMP-9,up-regulated the expression of claudin-5, but the trend is unstable (P>0.05).Conclusions: This work shows that the expression of NOD2, RIP2, NF-κB and MMP-9is significantly up-regulated and the expression of claudin-5has a markedly shrinkage after ischemia. Systemic administration ofnaringenin is effective which can ameliorate the neurological deficit, decreasethe infarct size and improve the brain edema caused by pMCAO. These effectsmay be through down-regulation of NOD2, RIP2, NF-κB and MMP-9andup-regulation claudin-5expression.