Protective Effect Of Cryptotanshinone Against Experimentally Induced Cerebral Ischemic Damage In Mice And Its Mechanism

Objective: Ischemic stroke with gradually increasing mortality is a majorcause of death and permanent disability worldwide. The pathologicalmechanism for cerebral ischemic damage is a complex perplexing cascadereaction, which involves inflammation, oxidative stress, increasedconcentrations of intracellular calcium, intracellular acidosis and apoptosis.Inflammation damage has been confirmed to play an important role in thepathogenesis of brain damage secondary to cerebral ischemia. Developinganti-inflammation therapies may bring progress in clinical treatment ofcerebral ischemia.Growing attention has been paid to traditional medicines, as they havebeen proved therapeutically/prophylactically fruitful against multiple diseases.Our previous study evaluated the protective effect of Tanshinone II A (Tan II A)against cerebral ischemia-induced inflammatory damage in an experimentalcerebral ischemia model. The promising results of Tan II A prompted us toinvestigate the neuroprotective efficacy of cryptotanshinone (CTs), which hasa chemical structure similar to that of Tan II A, against cerebral ischemicdamage. Cryptotanshinone derived from the roots of Salvia miltiorrhiza Bgeand Salvia przewalskii Maxim is the major active component and possessessignificant anti-inflammatory property just the same as Tan II A. Some studiesreported that CTs suppressed inflammatory cytokines through inhibition of theMAPK Signaling Pathways.As the classical and most extensively characterized MAPK pathway,extracellular signal-regulated kinase1and2(ERK1/2) pathway containsimportant mediators of the signal transduction responsible for cell growth andproliferation, and maybe potentially important signaling events in the inflammatory response. Several studies have shown an involvement of theERK1/2signaling pathway in focal cerebral ischemia, and inhibitors towardsthis pathway are able to diminish the ischemic area and exert a protectiveeffect by down-regulating the pro-inflammatory response after stroke.Upstream activated signals transfer phosphate to both threonine andtyrosine residues on ERK1/2. Then phospho-ERK1/2translocate into thenucleus through the nuclear membrane and activate a variety of transcriptionfactors such as Elk-1. Elk-1binds to and trans-activates genes including c-fos,a component of the transcriptional factor activator protein-1(AP-1), which canregulate the expression of many pro-inflammatory proteins. Finally,inflammatory factors such as IL-1β and TNF-α increase.The present study aimed to examine the neuroprotective role of CTs inischemic stroke, observe the expressions of phospho-ERK1/2, phospho-Elk-1, IL-1β and claudin-5in mice model of transient focal cerebral ischemiaand explore the underlying mechanism of the neuroprotection.Methods:Male, healthy CD1mice were subjected to modified transient middlecerebral artery occlusion (tMCAO), as described by Longa previously.Experiment1was used to evaluate the neuroprotection of CTs. Mice wererandomly assigned to six groups: tMCAO (transient middle cerebral arteryocclusion), Vehicle (tMCAO+1%Tween-80), CTs-L (tMCAO+CTs10mg/kg), CTs-M (tMCAO+CTs20mg/kg) and CTs-H (tMCAO+CTs40mg/kg) and Sham operated group.The drugs were administrated by intraperitoneal injection according todifferent group after one hour of cerebral ischemia and twenty-four hours ofreperfusion. Sham operated group and tMCAO group received equal volumedistilled water. At24h after reperfusion, neurological deficit was evaluated asdescribed by Connolly. Infarct volume was analyzed with2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain water ontent wasmeasured by wet-dry method.Experiment2was used to detect CTs’ influence on ERK1/2activation signaling pathway and blood brain barrier, investigating the potentialmechanism of the neuroprotective effect of CTs in cerebral ischemia. Micewere randomly assigned to three groups: Sham operated group, Vehicle(tMCAO+1%Tween-80) and CTs-H (tMCAO+CTs40mg/kg) group.Immunohistochemistry, western blot, immunofluorescence and RT-qPCR(Real-time reverse transcription-quantitative PCR) were used to analyze theexpression of p-ERK1/2, p-Elk1, IL-1β and claudin-5. Evans Blue dyeextravasation was explored at24h after reperfusion.Results:1. CTs reduced neurological deficits. Mice in tMCAO、Vehicle、CTs-Lgroup, CTs-M group and CTs-H group performed a left palsy. Compared withVehicle group, the neurological deficit scores in CTs-H group weresignificantly reduced (P <0.05). The neurological deficit scores were alsoreduced in CTs-L group and CTs-M group. However, no significant differencewas found among Vehicle group, CTs-L group and CTs-M group (P>0.05).2. CTs reduced the infarct volume. No infarction was measured inSham-operated group. Compared with Vehicle group, the infarct volume waslessened in CTs-L group, CTs-M group and CTs-H group(CTs-L%HLV:39.42%±0.76%; CTs-M%HLV:37.65%±1.12%; CTs-H%HLV:32.53%±0.33%, P <0.05). At CTs-H group, CTs reduced the infarct volume moresignificantly than CTs-L group and CTs-M group (P <0.05).3. CTs reduced the brain water content. Compared with Vehicle group, allthree administration groups showed decline in the brain water content(CTs-Hvs. Vehicle:80.98%±0.98%vs.83.48%±0.49%, P <0.05; CTs-M vs.Vehicle:81.64%±0.64%vs.83.48%±0.49%, P <0.05; CTs-L vs. Vehicle:82.36%±1.62%vs.83.48%±0.49%, P <0.05). However, there was nosignificant difference for the effect of CTs on the brain water content amongCTs-L group, CTs-M group and CTs-H group.4. CTs down-regulated the expression of p-ERK1/2, p-Elk1, IL-1β.Outcome of immunohistochemistry showed that the number of positive cellsof p-ERK1/2and p-Elk1dramatically increased in ischemic cortex around infarct regions of Vehicle group at24h after reperfusion. CTs (40mg/kg)significantly reduced the positive cells of p-ERK1/2and p-Elk1after tMCAO.Consistent with the results of immunohistochemistry, Western blottinganalysis showed that CTs (40mg/kg) significantly downregulated theexpression of p-ERK1/2and p-Elk1(P <0.05). Confocal microscope alsoshowed that CTs decreased the expression of p-Elk1. Simultaneously,RT-qPCR analysis showed a significant decrease of IL-1β in CTs-H group (P<0.05).5. CTs protected the integrity of BBB. Marked extravasation of EB intothe cerebral parenchyma was detected in the ischemic hemisphere in mice,indicating severe disruption of the BBB after brain ischemia. CTs (40mg/kg)administered immediately after reperfusion substantially reduced EB leakagevolume (P <0.05). The expression of claudin-5significantly decreased afterreperfusion. Compared with Vehicle group, claudin-5’s expression wasincreased by western blot and RT-qPCR at24h in CT-H groups (P <0.05).Conclusions：Early and short-time administration of CTs in tMCAOmice can decrease neurologic impairment, reduce the infarct volume and thebrain water content, down-regulate the expression of p-ERK1/2, p-Elk1andIL-1β, up-regulate the expression of claudin-5and ameliorate theextravasation of EB into the cerebral parenchyma. In conclusion, our studyshowed that early administration of CTs elicit the neuroprotective effect in adose-dependent manner, which might be associated with the down-regulationof p-ERK1/2, p-Elk1and IL-1β expression, up-regulation of claudin-5expression, inhibition of the inflammatory response as well as amelioration ofBBB permeability in cerebral ischemia.