Anti-apoptosis Activiaty Of Silibinin Is Mediated By EGFR/ERK Pathway In Permanent Cerebral Ischemia

Objective: Cerebral ischemia has been the capital issue for disability in China.The fatal injury following after ischemia in turn aggravates brain ischemia,including inflammation,oxidative stress,cell apoptosis and the releasing of cell kinase,free base and excited toxic substances.Apoptosis is a kind of programmed cell death in cerebral ischemia process,and it is active cell death process and it is also an important mechanism of neuron death.Apoptosis plays a important role in cerebral ischemia.Silibinin is a kind of mixture of polyphenolic flavonoids,which has been confirmed to elicit a variety of biological effects on anti-inflammation,anti-oxidant stress,regulating mitochondria and anti-apoptosis,which are the reasons why it can be used in hepatitis,cancer.Meanwhile,epidermal growth factor receptor(EGFR)belongs to Epidermal Growth Factor Receptor Family.EGFR widely exists in the surface of epithelial cell,fibroblast,gliocyte,Keratinocyte of mammal.EGFR acts as an important role for cell in growing,proliferation and differentiation.Recent studies have shown that silibinin can influence the expression of EGFR in order to affect cell proliferation and apoptosis.But there was few research ever found to investigate the nerve protective effect and its mechanism of silibinin in the acute phase of cerebral ischemia.Our experiment established a permanent middle cerebral artery occlusion model in mice successfully.We observed the protective activity of silibinin in cerebral ischemia and its effect on apoptosis and EGFR/ERK signaling pathway.Methods: This study was comprised by two steps: Experiment 1 was used to investigate silibinin's neuroprotective effect in ischemia brain;Experiment 2 was intended to evaluate the expression of p-EGFR/EGFR,p-ERK/ERK,cleaved-caspase3,Bcl-2 and Bax by western blotting and immunohistochemistry.Silibinin(Zelang Medical Technology Co.,Ltd.,Nanjing,China)with purity of more than 98% dissolved in 0.9% NaCl.Mice were administered with silibinin at three doses of 50,100 and 200 mg/kg intragastrically 30 min before pMCAO.Mice were reanesthetized and killed 24 h(n1)and 72 h(n2)after pMCAO.At each time points,mice were divided into 5 groups randomly(n1 = n2 = 30,6 mice in each group).Group 1: mice as the sham-operated group that received equal volume saline(Sham);Group2: MCAO controls that MCAO-operated received equal volume 0.9% saline(pMCAO);Group 3: mice that MCAO-operated received silibinin at 50 mg/kg(Silibinin-L);and Group 4: mice MCAO-operated that received silibinin at 100 mg/kg(Silibinin-M);Group 5: mice that received silibinin at 200 mg/kg(Silibinin-H).Infarct volume and water content were evaluated 24 h after pMCAO.Nissl staining,western blotting and immunochemistry was evaluated 24 h and 72 h after pMCAO.Results:1 Brain ischemia volume was measured 24 h after MCAO by TTC.In MCAO,an extensive lesion was developed in both striatum and cortex(Fig.1A).The infarct volume was significantly lessened from 59.28% ± 2.92% in MCAO group to 43.08% ± 5.14% in Silibinin-M group and 43.77% ± 5.05% in the Silibinin-H group(P < 0.05,Fig.1B).However there was no difference between Silibinin-L group and MCAO group.The data suggested obviously that Silibinin in Middle and High dose group acted a neuroprotective role in cerebral ischemia at different doses in infarct volume.2 Wet-dry method was meant to measure both hemispheres brain water content.Brain edema in the ipsilateral and contralateral hemisphere of each treatment group was shown in Fig.2.There was no significant difference in contralateral hemisphere of brain water content in the MCAO group compared with the three silibinin groups(P > 0.05 for all).The three doses of Silibinin declined the percentage of brain water content in ipsilateral hemispheres after stroke.Compared with MCAO group,middle dose of Silibinin reduced the brain water content of ipsilateral hemispheres significantly(pMCAO vs.Silibinin-M: 86.94% ± 0.61% vs.84.81% ± 0.79%,P < 0.05,Fig.2).In low and high groups,the brain water content was reduced(pMCAO vs.Silibinin-L and Silibinin-H: 86.94% ± 0.61% vs.86.11% ± 0.48% and 86.93% ± 0.38%,P > 0.05,but did not make significant difference with MCAO group.3 The coronal brain sections for Nissl staining from the Sham,MCAO and the Silibinin-M treated mice 24 h and 72 h after the injury is presented.Neurons with abundant nissl bodies were significantly decreased in the pMCAO compared with the Silibinin-M group,which was significantly restored by silibinin administration(P < 0.05).4 The expression of p-EGFR/EGFR,p-ERK/ERK,cleaved-caspase3,Bax and Bcl2 were regulated by silibinin: After cerebral ischemia,silibinin were employed to investigate the regulation of p-EGFR/EGFR,p-ERK/ERK,cleaved-caspase3,Bax and Bcl2 expression at 24 h and 72 h after MCAO at the level of western blotting(Fig.4 and Fig.5)and immunohistochemical staining(Fig.6 and Fig.7).Western blotting 24 h after MCAO(Fig.3A.B)revealed that p-EGFR/EGFR,p-ERK/ERK,Bcl2 decreased,while cleaved-caspase3,Bax increased after MCAO.P-EGFR/EGFR increased in Silibinin-M and Silibinin-H group compared with MCAO,obviously(P < 0.05).Compared with MCAO,p-ERK/ERK and Bcl2 were up-regulated only in Silibinin-M group(P < 0.05).On the other hand,cleaved-caspase3 and Bax were down-regulated in all the three doses of silibinin groups(P < 0.05).72 h later after MCAO(Fig.4.A.B)excluded that p-EGFR/EGFR and Bcl2 enhanced only in Silibinin-M group,p-ERK/ERK was promoted in all groups administered silibinin(P < 0.05).Cleaved-caspase3 declined in all groups administered silibinin(P < 0.05),while Bax in Silibinin-M and Silibinin-H group(P < 0.05).5 Immunohistochemistry: Consistent with the results of western blotting,immunohistochemistry analysis also showed that the positive cells of p-ERK,p-EGFR were up-regulated,while the the positive cells of cleaved-caspase3 was down-regulated in drug group,compared with that in MCAO group(Fig.6A and Fig.7A).Middle dose silibinin significantly enhanced the expressions of p-ERK,p-EGFR,and eliminated cleaved-caspase3 both 24 h and 72 h after MCAO(P < 0.05,Fig.6.B.and Fig.7.B.).Conclusions: Our result showed that silibinin significantly reduced infarct volume and brain edema,which were accompanied by up-regulation of Bcl-2,p-ERK/ERK,p-EGFR/EGFR and down-regulation of Bax,cleaved-caspase3 in ischemic brain tissue after stroke.We conducted that silibinin might exert anti-apoptotic effects on ischemic brain through activating EGFR/ERK signaling pathway.