The Expression Of PAkt,pmTOR,HIF-1α In Cerebral Ischemia In Rats And The Neuroprotective Effect Of Silibinin

Objective: Cerebral ischemia is the most common type of cerebralvascular disease with serious disability and high mortality. The pathologicalmechanism for ischemic injury was a complex cascade reaction, includinginflammation, apoptosis, oxidative stress and perturbation of calciumhomeostasis. It is commonlly believed that excessive immunological reactionof inflammation exist in ischemic and necrosis region, and then result ininflammatory injury. Therefore, it is one of the main ways to treat acutecerebral infarction by alleviating inflammatory injury. Apoptosis regulatingmechanism is not very clear, but recent experimental studies and clinicalobservations: the balance of anti-apoptotic protein Bcl-2and the proapoptoticprotein Bax plays a key role in the regulation of apoptosis. Currently,anti-inflammatory and anti-apoptotic treatment in cerebral infarction did notachieve the ideal desired effect. Looking for the initiating factor for a cascadeof upstream and drug intervention may be a new target for the treatment ofcerebral ischemia. Akt/mTOR/HIF-1α signal pathway plays an important rolein the process of cell proliferation, growth, differentiation, and apoptosis byregulating the cell cycle, protein synthesis and degradation, energymetabolism, and a variety of ways. Akt/mTOR/HIF-1α signal pathwayinduced NF-κB (nuclear factor kappa B) activation pathway and Bcl-2andBax balance adjustment play an important role in nerve cells inflammatoryresponses and apoptosis of ischemic cerebrovascular disease. Silibinin is alignan compound of the extract obtained from the medicinal plants of theAsteraceae milk thistle seed testa called flavonoids, abundant ofpharmacological and clinical studies find that silibinin has anti-inflammatory,anti-apoptosis and anti-tumor effects, but its mechanism underlying is poorlyunderstood.The purpose of this experiment is to evaluate the potential neuroprotective effect of silibinin and underlying mechanisms after cerebralischemia induced in male adult Sprague-Dawly rats by permanent middlecerebral artery occlusion (MCAO).Methods: Male, healthy Sprague-Dawley rats were used and randomlyassigned to five groups: Sham operated group(Sham), stroke with vehiclegroup (Vehicle), stroke with high-dose (100mg/kg per day) silibinin group(Silibinin-H), stroke with low-dose (50mg/kg per day) silibinin group(Silibinin-L) and normal control group (Normal). MCAO model was inducedby using intraluminal filament technique in rats. Silibinin (50,100mg/kg) wasintragastric administrated30minutes before cerebral ischemia, then once dailyon the following days. For Vehicle, Sham and Normal group, equal volumesaline was administered in the same manner. Neurological behavior wasevaluated at24h and72h after operation then rats were sacrificed. Brainwater content was measured by wet-dry method; Infarct volume was analyzedwith2,3,5-triphenyltetrazolium chloride (TTC) staining at24h; Akt,phosphorylated Akt (pAkt),mTOR,phosphorylated mTOR (pmTOR),HIF-1α,Bcl-2,Bax,NF-κB and claudin-5expressions were measured byimmunohistochemistry, Western Blot, RT-PCR.Results:1Rats in Sham group had a neurological score of zero at all time points.Rats in Vehicle group, silibinin high dose group and low dose groupperformed a left palsy. Neurological deficit score in high dose group wassignificantly decreased compared with Vehicle group (P <0.05). There was nosignificant difference in the neurological deficit score between Vehicle groupand low dose group (P>0.05).2In Silibinin-L group the brain water content reduced significantlycompared with Vehicle group at72h (P <0.05). Compared with Vehiclegroup, high dose of silibinin reduced the brain water content of ipsilateralhemispheres significantly at both24h and72h (P <0.05).3Infarct size was decreased in Silibinin-H group compared with that inVehicle group at24h (P <0.05), but no statistical significance was observed between Silibinin-L group and Vehicle group (P>0.05).4Compared with normal-control group, the protein levels of pAkt,pmTOR and HIF-1α, and the mRNA levels of Akt and mTOR wereup-regulated at24h and72h after pMCAO (P <0.05).The results ofimmunohistochemistry of pAkt and pmTOR were coincident with those ofRT-PCR and Western blot.The expression of HIF-1α in immunohistochemistrywas coincident with western blot, but not affected at the mRNA level (P>0.05).5Silibinin promoted the expression of pAkt, pmTOR and HIF-1α afterpMCAO. Immunohistochemistry showed that the expression of pAkt, pmTORand HIF-1α were up-regulated at24h and72h after cerebral ischemia treatedwith silibinin. In Sham group, few cells stained by pAkt, pmTOR and HIF-1αin the cortex. In Vehicle group, the number of cells stained by pAkt, pmTORand HIF-1α was increased in the ischemic cortex, and pmTOR expressed bothat cytoplasm and nucleus. Compared with Vehicle group, the number of cellslabeled with pAkt, pmTOR and HIF-1α in Silibinin-H group was significantlyincreased (P <0.05). However, there were no significant differences betweenVehicle group and Silibinin-L group. In agreement with the results ofimmunohistochemistry, western blotting and RT-PCR also showed asignificant increase of pAkt, pmTOR, HIF-1α and Akt, mTOR respectively inSilibinin-H group at protein and mRNA levels.6Compared with Vehicle group, the expression of Bcl-2was upregulatedand the Bax, NF-κB was downregulated in Silibinin-H group at protein level(P <0.05). However, there were no significant differences between Vehiclegroup and Silibinin-L group. In agreement with the results of Western blotting,RT-PCR showed the same result about Bcl-2, Bax and NF-κB.7Silibinin ameliorated BBB permeability. Western Blot and RT-PCRshowed that Silibinin-H upregulated the expression of claudin-5in thecerebral ischemia (P <0.05). But no statistical significance was observed insilibinin low dose group.Conclusions: Our study showed that the expressions of pAkt, pmTOR and HIF-1α were up-regulated after cerebral ischemia. Systemic administration ofhigh dose silibinin is effective which can decrease neurologic impairment andtissue injury, improve the brain edema and decrease the infarct size undercerebral ischemic conditions, and this effect may be through up-regulation ofpAkt, pmTOR, HIF-1α, Bcl-2, down-regulation of Bax, NF-κB, thusinhibiting the inflammation and anti-apoptosis; up-regulation of claudin-5expression to ameliorated BBB permeability.