| Objective: Stroke is the most frequent cause of permanent disability inadults and the third leading cause of death worldwide. Despite considerableadvances in the understanding of the pathophysiology of acute cerebralischemia, therapeutic options for acute ischemia are still limited. Berberine(BBR), an alkaloid derivative from Berberis Vulgaris L, has been usedextensively in traditional Chinese medicine to treat diarrhea and diabetes.Previous studies have reported that BBR has many beneficial biologicaleffects, including immunomodulation, anti-diabetic metabolic effect,anti-inflammatory effect, and anti-tumor activity. Furthermore, studies haveshown that BBR exerts a protective effect on several models of cerebralischemic injury and myocardium injury induced by ischemia/reperfusion.However, the functional role of BBR in acute phase of cerebral ischemia is notclear so far.After ischemia, depending on the severity of the insult and the ability ofthe cell to maintain adenosine triphosphate (ATP) synthesis, there are mainlytwo forms of neuron death, necrosis and apoptosis. The serine-threoninekinase, Akt, which is also known as protein kinase B, plays an important rolein the cell death/survival pathway. Akt phosphorylation is neuroprotectiveagainst ischemic injury. Activated Akt phosphorylates several downstreamtargets of the PI(3)K pathway, including the constitutively activeserine-threonine kinase glycogen synthase kinase3-β (GSK3β)(Ser9). GSK3βis involved in fundamental cellular processes that can determine cell death.GSK3β is particularly abundant in the central nervous system and isneuron-specific. Akt phosphorylates GSK3β on Ser9to render it inactive, a proposed mechanism by which neurons become resistant to apoptotic stimuli.GSK3β has also been reported to be involved in ischemic brain injury. GSK3βinhibition augments the binding of cAMP response element binding protein(CREB)(Ser133) and suppresses the binding of nuclear factor-kappa B(NF-κB) p65(Ser276) to the nuclear co-activator CREB binding protein (CBP)(Martin et al.,2005). GSK3β regulated the inflammatory response bydifferentially affecting the nuclear amounts of transcription factors NF-κBsubunit p65. The nuclear transcription factor CREB is known to regulate theexpression of a large number of genes in a variety of physiological contexts,such as proliferation, differentiation, growth, survival and adaptation of alltypes of cells. Since1990s, phosphorylated CREB (pCREB), activated form ofCREB, has been examined in several ischemia models. Cerebral ischemiacaused a burst of CREB phosphorylation and the corresponding expression ofcAMP-responsive element (CRE)-targeted genes which encodeneuroprotective molecules, such as the protein B cell lymphoma/lewkmia-2and the brain-derived neurotrophic factor in neuron. CREB is known to playimportant roles in synaptic plasticity, neurogenesis and axon growth afterischemia. Ample evidences have suggested that Akt/GSK signaling pathwayplays a central role in physical and pathological conditions to differentlyregulate inflammation factors as well as boost survival.The present project is to characterize the profound impact of BBR onoutcome after acute ischemic stroke, and identify its targets and protectivesignaling pathways in permanent middle cerebral artery occlusion (pMCAO)brain.Methods: This study included two parts: Experiment1was used todetect TSA’s neuroprotective effect in cerebral ischemia. Experiment2wasused to evaluate the expression of phospho-Akt, phospho-GSK3,phospho-CREB, claudin-5and NF-κB in the cerebral ischemia. Experiment1,ninety-six male Sprague-Dawley rats were randomly divided into four groups(24rats in each group): Sham operated group that received equal volume0.9%NaCl (Sham); pMCAO group that received equal volume0.9%NaCl after pMCAO (pMCAO); low dose group that received BBR at10mg/kg afterpMCAO (BBR-L); and high dose group that received BBR at40mg/kg afterpMCAO (BBR-H). pMCAO group were subjected to permanent focal cerebralischemia by right MCA occlusion, neurological deficit was evaluated using amodified six point scale24h after MCAO. Brain water content was measured;infarct size was analyzed with2,3,5-triphenyltetrazolium chloride (TTC).Experiment2, rats were randomly individed into four groups (24rats in eachgroup): Sham operated group that received equal volume0.9%NaCl (Sham);pMCAO group that received equal volume0.9%NaCl after pMCAO(pMCAO); low dose group that received BBR at10mg/kg after pMCAO(BBR-L); and high dose group that received BBR at40mg/kg after pMCAO(BBR-H). BBR solution (10mg/kg or40mg/kg) was injected intraperitoneallyimmediately after MCAO. Immunohistochemistry, Western blotting andRT-PCR were used to analyse the expression of phospho-Akt, phospho-GSK3,Results:1. Brain infarction volume: No infarction was observed in thesham-operated group. In pMCAO group, an extensive lesion was developed inlateral cortex (Fig.2A). The infarct volume was significantly reduced from44.17±7.44%in pMCAO group to34.73±2.63%in the BBR-L group (P <0.05) and30.93±6.66%in the BBR-H group (P <0.01)(Fig.2A and2C)(n=6in each group).2. Neurological deficit was examined and scored on a5-point scale andfor statistical analysis the Mann-Whitney U-tests were conducted. Theneurological deficits scores were significantly higher in pMCAO group (3.50±0.89) compared with Sham control. Berberine decreased these scores inBBR-L (3.13±0.68) and BBR-H (2.88±0.85) group (10mg/kg vs. pMCAOgroup, P <0.05;40mg/kg vs. pMCAO group, P <0.01; Fig2B). And there isno significant difference between BBR-L group and BBR-H group (10mg/kgvs.40mg/kg, P>0.05).3. Brain water content measurement: Ipsilateral brain water content ofSham group was78.59±1.31%. Compared with pMCAO control, BBR-L and BBR-H group showed an intense decline in the percentage of brain watercontent (pMCAO group vs. BBR-L group:84.35±1.69%vs.82.44±1.83%, P<0.05; pMCAO group vs. BBR-H group:84.35±1.69%vs.81.84±1.91%, P <0.05)(Fig.2D)(n=6in each group).5. TSA’s role to pAkt, pGSK3β, pCREB and claudin-5and downregulatedthe expression of NF-κB (p65) expression: The expression of pAkt, pGSK3β,pCREB and claudin-5were upregulated at protein level after systemicadministration of BBR in ischemia brain. The localization of pAkt, pGSK3β,pCREB protein was identified by immunohistochemistry at24h afterpMCAO. Representative immunohistochemistry photographs for pAkt andNF-κB were exhibited in Fig.3A. As shown in Fig.3B and C the expressionsof pAkt, pGSK3β and pCREB in BBR-L and BBR-H group (n=6in eachgroup) was significantly increased at24h after surgery vs. pMCAO (P <0.01).In agreement with results of immunohistochemistry, western blotting analyses(Fig.4A, B) also showed a significant increase of pAkt, pGSK3β and pCREBin BBR-L and BBR-H group (n=6in each group) vs. pMCAO (P <0.01).The expression of NF-κB (p65) was upregulated after ischemia, and wasdownregulated significantly at mRNA and protein level after systemicadministration of BBR in ischemia brain. In sham operated animals, few cellsstained by NF-κB (p65) were seen in the cortex (Fig.3A5).24h after theinjury, an intense staining of NF-κB was observed at both cytoplasm andnucleus in the ischemic cortex (Fig.3A6). In both BBR-L and BBR-H group,the number of cells labeled with NF-κB (p65) was declined significantly vs.pMCAO (P <0.01)(Fig.3A7, A8). Compared with pMCAO group, thenuclear NF-κB was significantly decreased in BBR-L and BBR-H group (bothP <0.01) by immunohistochemistry (Fig.3A, C). In agreement with theresults of western blotting (Fig.4C, D) and RT-PCR (Fig.5A, B), theexpression of NF-κB was significantly decreased in BBR-L and BBR-H groupvs. pMCAO group (both P <0.01).Conclusions: In conclusion, our study provides beneficial evidences forearly administration of BBR and its underling mechanisms after acute brain ischemia. BBR protected the brain from damage caused by MCAO, BBRlessened neurological deficit scores, the brain water content and infarct size.Specifically, the protective effect of BBR against ischemic injury may throughAkt/GSK signaling activation, upregulation of pCREB, downregulation ofNF-κB nuclear transposition and ameliorated BBB permeability. So BBRshould be one of the strategic targets for cerebral ischemic therapies. |
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