The Neuroprotective Effect Of Cepharanthine And The Underlying Mechanism In A Mouse Model Of Focal Cerebral Ischemia

Stroke is the major disease,which threatens human health in China. So far, stroke is a leading cause of mortality, of which ischemic stroke accounts for approximately 80–85%. According to the World Health Organization, the morbidity of Chinese cerebrovascular disease ranks first in the world, which is about 1 times higher than that of America’s. 40% of stroke patients not expected to recover independence. In the victims of ischaemic stroke, approximately 75% of all cases are left with long-term disability to varying degrees, 40% of which have severe disability, leading to aggravating healthy quality of life. What’s more, because of its high morbidity, disability, recurrence rate, and the expenses of the therapy, ischemic stroke brings a heavy burden to the social and family.Secondary cerebral injury is the key factor for aggravation. How to alleviate the secondary cerebral injury is the mayor project. The pathological mechanisms that trigger ischemic brain damage are a rapid cascade reaction which includes the release of excitatory amion acids, oxidative stress, inflammation, apoptosis, and so on. Treatment with recombinant tissue-plasminogen activator(rt-PA), a thrombolytic agent, is the only FDA-approved therapy for acute stroke but it is limited to a small proportion of patients because of its narrow time window(3-6 h) and risk of intracranial hemorrhage and brain edema. This underscores the need for new therapeutic avenues to stroke treatment. It is a focus in neuroscience that looking for the ideal neuroprotective agents that can block ischemic cascade reaction. Chinese herbal medicines have low side effects and no safety tissue, which is good source.Cepharanthine(CEP) is a natural alkaloid extracted from the plant Stephania cepharantha Hayata. It is approved by the Japanese Ministry of Health for the treatment of radiation-induced leucopenia. The best known effect of CEP is anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory properties,and so on. Several in vivo and in vitro studies have demonstrated that CEP inhibits tumor necrosis factor(TNF)-α-mediated nuclear factor-κB(NF-κB) stimulation, platelet aggregation, and suppresses cytokine production. It has also been shown to have a protective effect against renal ischemia-reperfusion injury in rats. No safety issues have been observed with CEP, and side effects are very rarely reported.Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(MCAO), as described by Longa previously. CEP was administered(i.p.) in the mouse model of MCAO. The neuroprotection of CEP was analysed by evaluating neurological deficit scores, brain edema, infarct volume and blood-brain barrier leakage. The anti-inflammatory effect of CEP was analyzed by levels of p38MAPK(mitogen-activated protein kinases), JNK(c-jun N-terminal kinase), NF-κB and NLRP3, IL-1β. The possible mechanisms of its neuroprotection were discussed. PartⅠ The neuroprotective effect of cepharanthine in the mice of focal cerebral ischemiaObjective: The aim was to study the neuroprotective effects of cepharanthine by evaluating the neurological deficit scores and infarct volume.Methods: Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(MCAO). The mice were randomly divided into 5 groups randomly. Group 1: Sham-operated group(Sham) that received equal volume normal saline; group 2: MCAO group(MCAO) that received equal volume normal saline after MCAO; group 3: CEP low dose(CEP-10) mice that received CEP at 10 mg/kg after MCAO immediately after cerebral ischemia by intraperitoneal injection, then once daily thereafter; group 4: CEP middle dose(CEP-20) mice that received CEP at 20 mg/kg after MCAO immediately after cerebral ischemia by intraperitoneal injection, then once daily thereafter; group 5: CEP high dose(CEP-40) mice that received CEP at 40 mg/kg after MCAO immediately after cerebral ischemia by intraperitoneal injection, then once daily thereafter. Neurological behavior evaluated by neurological deficit scores, and infarct volume analyzed with 2, 3, 5- triphenyltetrazolium chloride(TTC) were evaluated at 24 h and 72 h after MCAO to estimate cepharanthine neuroprotective effect.Results:1 Mice in Sham group had no palsy and a neurological score of zero. Mice in MCAO, CEP-10, CEP-20 and CEP-40 group performed a left palsy. Compared with MCAO group, there was a significant improvement in neurological deficit scores in CEP-20 and CEP-40 group at 24 h and 72 h after MCAO(P < 0.05). Although CEP-10 group improve neurological deficit scores, there was no significant effect in CEP-10 group compared with MCAO group at both 24 h and 72 h after MCAO(P > 0.05 for all).2 Compared with MCAO group, CEP-10 group had no effect on infarct volume both at 24 h and 72 h(P > 0.05). CEP-20 and CEP-40 group reduced the infarct volume significantly both at 24 h and 72 h(P < 0.05). Based on the results above, we demonstrated that cepharanthine administered at middle dose(20 mg/kg) and high dose(40 mg/kg) per day have a better therapeutic effect after stroke, and therefore we focused on the cepharanthine treatment at middle dose(20 mg/kg) per day for the subsequent study.Conclusions: Administration of cepharanthine is effective which can ameliorate the neurological deficit scores and decrease the infarct size. Part Ⅱ The effect of cepharanthine on blood-brain barrier and the underlying mechanism in the mice of focal cerebral ischemiaObjective: The aim is to explore the effect of cepharanthine on blood-brain barrier and the underlying mechanism.Methods: Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(MCAO). The mice were randomly divided into 3 groups randomly. Group 1: Sham-operated group(Sham) that received equal volume normal saline; group 2: MCAO group(MCAO) that received equal volume normal saline after MCAO; group 3: CEP middle dose(CEP-20) mice that received CEP at 20 mg/kg after MCAO immediately after cerebral ischemia by intraperitoneal injection, then once daily thereafter. Blood-brain barrier leakage, measured using Evans blue extravasation, was evaluated at 24 h after MCAO to estimate cepharanthine’s effect on blood-brain barrier. Wet-dry method was used index to measure brain edema. Western-blotting and q RT-PCR were used to analyse the ZO-1, occluding and claudin-5 expression in the ischemic cortex.Results:1 Compared with MCAO group, CEP-20 group reduced the brain water content significantly both at 24 h and 72 h(P < 0.05).2 An extensive BBB disruption was found in animals from the MCAO group. The protective effects of CEP were also observed by examination of Evans blue content at 24 h. As compared with the MCAO group, significantly lower Evans blue extravasation was observed in the CEP-20 groups(P < 0.05 vs. MCAO).3 In western-blotting analysis, compared with MCAO group, the ZO-1, occluding and claudin-5 protein levels in the right cortex were significantly increased in CEP-20 group at 24 h and 72 h after cerebral ischemia(P < 0.05).4 In agreement with the results of western-blotting, compared with MCAO group, ZO-1, occluding and claudin-5 m RNA levels in the right cortex were significantly increased in CEP-20 group at 24 h and 72 h after cerebral ischemia(P < 0.05).Conclusions: ZO-1, occluding and claudin-5 expressions were significantly decreased and all these effects were improved by cepharanthine at 24 h and 72 h after focal cerebral ischemia. CEP reduced the brain edema and improved the blood-brain barrier permeability, suggesting that cepharanthine had a good effect on the blood-brain barrier. This is in accordance with the neuroprotective effect of cepharanthine in the mice of focal cerebral ischemia. Therefore, we estimated that cepharanthine might protect blood-brain barrier by up-regulated ZO-1, occluding and claudin-5 expressions. Part Ⅲ Anti-inflammatory Effect of cepharanthine in focal cerebral ischemia injury in mice and its mechanism of signal transduction pathwayObjective: The aim is to estimate the anti-inflammatory effects of cepharanthine in focal cerebral ischemia injury in mice and to explore its possible mechanisms and related signal transduction pathways.Methods: Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(MCAO). The mice were randomly divided into 3 groups randomly. Group 1: Sham-operated group(Sham) that received equal volume normal saline; group 2: MCAO group(MCAO) that received equal volume normal saline after MCAO; group 3: CEP middle dose(CEP-20) mice that received CEP at 20 mg/kg after MCAO immediately after cerebral ischemia by intraperitoneal injection, then once daily thereafter. Immunohistochemistry, Western-blotting and q RT-PCR were used to analyze the expressions of NF-κB, p38 MAPK, JNK, NLRP3 and IL-1β in the ischemic cortex at 24 and 72 h after MCAO. Confocal microscopy was used to analyze the expression of NF-κB, NLRP3 and IL-1β in the ischemic cortex at 24 h after MCAO.Results:1 In immunohistochemistry, compared with MCAO group, the number of positive cells of NF-κB, phospho-p38 MAPK, phospho-JNK, NLRP3 and IL-1β were significantly decreased in CEP-20 group at 24 h and 72 h after cerebral ischemia(P < 0.05).2 NLRP3, IL-1β and NF-κB expressions were detected in the neurons. The expressions of NLRP3, IL-1β and NF-κB were up-regulated and NF-κB translocated into nuclei. Compared with MCAO group, CEP-20 group decreased the numbers of NLRP3, IL-1β and nuclei NF-κB at 72 h after cerebral ischemia.3 In western-blotting analysis, compared with MCAO group, the expressions of NF-κB, phospho-p38 MAPK, phospho-JNK, NLRP3 and IL-1β in the right cortex were significantly decreased in CEP-20 group at 24 h and 72 h after cerebral ischemia(P < 0.05).4 In agreement with the results of immunohistochemistry and western-blotting, compared with MCAO group, the m RNA levels of NF-κB, NLRP3 and IL-1β in the right cortex were significantly decreased in CEP-20 group at 24 h and 72 h after cerebral ischemia(P < 0.05).Conclusions: We have demonstrated that cepharanthine could protect against cerebral ischemia injury and suppress the expression of NF-κB, p38 MAPK, JNK, NALP3 and IL-1β in the ischemic cortex, suggesting that cepharanthine might exert anti-inflammatory and neuroprotective effects by suppressing NLRP3, IL-1β and down-regulating NF-κB and p38 MAPK, JNK-associated signaling pathway.