The Neuroprotective Effect Of Celastrol And The Underlying Mechanism In Experimental Ischemic Stroke

Objective: Ischemic stroke with high mortality and serious disability is oneof the three leading causes of death in the adults world. The pathologicalmechanism for ischemic injury was a perplexing cascade reaction, includinginflammation, oxidative stress, calcium overload and glutamate excitotoxicity.Excessive immunological reaction of inflammation and oxidative stress innecrosis and ischemic region have been confirmed to remain a major cause inthe pathogenesis of brain injury secondary to ischemia. Oxidative stress due toreactive oxygen and nitrogen species can bring about lipid peroxidation,protein oxidation, and DNA damage in brain ischemia/reperfusion injury,which will further aggravate the brain damage. Therefore, it is a main measureto treat acute cerebral infarction by alleviating inflammatory and oxidativestress damage. c-Jun N-terminal kinases (JNKs), known as stress-activatedprotein kinases (SAPKs) represents an important sub-family of the MAPkinase group, which Participate in the pathophysiologic progress of cerebralischemia. The activated signaling pathway of NF-κB plays an important rolein cerebral ischemia. Despite numerous therapeutic trials, stroke is still themajor cause of death in the world and currently lacks proper and effectivetreatment. Looking for new inhibitors of the initiation of inflammation andoxidative stress may bring progress in treatment of cerebral ischemia.Celastrol has been proved to elicit a vanity of biological effects through itsanti-oxidant, anti-inflammatory properties The therapeutic potential ofcelastrol has been tested invarious inflammatory disease models Alzheimer’sdisease, systemic lupus erythematosus, and rheumatoid arthritis. However,little is known regarding the effect of celastrol in the acute phase of ischemicstroke. Our experiment observed the expressions of p-JNK, p-c-Jun and NF-κB in rat model of focal cerebral ischemia; investigated the neuroprotective effect of Celastrol and his effect may be through downregulation of theexpression of p-JNK, p-c-Jun and NF-κB.Methods: Male, healthy Sprague-Dawley rats were used and randomlyassigned to four groups: Sham operated group（Sham）, Vehicle group,Celastrol high dose (MCAO+Celastrol3mg/kg, Cel-H), Celastrol low dose(MCAO+Celastrol2mg/kg, Cel-L) group. MCAO model was induced byusing intraluminal filament technique in rats. Celastrol (2,3mg/kg) wasadministrated by intraperitoneal injection after cerebral ischemia. For MCAO,Sham and Normal group, equal volume saline was administered in the samemanner. We used a pMCAO model and administered celastrolintraperitoneally immediately after cerebral ischemia. At24h after stroke,neurological deficit, brain water content, and infarct size were measured.C-Jun, nuclear factor-κB (NF-κB) were measured by reverse transcriptionpolymerase chain reaction (RT-PCR), immunohistochemistry (IHC), andWestern blot. Phospho-JNK and Phospho-c-Jun were measured by IHC, andWestern blot.Results:1Rats in Sham group had a neurological score of zero at24h. Rats inVehicle group, high dose group and low dose group performed a right palsy.Compared with Vehicle group, the neurologic deficit scores in Cel-H groupwere significantly reduced (P <0.05), but no significant difference was foundbetween vehicle group and Cel-L group (P>0.05).2No infarction was measured in Sham-operated group, while extensivelesion was developed in both striatum and lateral cortex in vehicle controlgroup. Comparedwith Cel-L group, Cel-H group showed intenser decline inthe infarct volume (Cel-H vs. Vehicle:32.55±2.99%vs.42.26±1.92%, P <0.05; Cel-L vs. Vehicle:38.99±2.20%vs.42.26±1.92%, P <0.05).Moreover, there was a significant difference for the effect of celastrol oninfarct volume between Cel-L group and Cel-H group (Cel-H vs. Cel-L:32.55±2.99%vs.38.99±2.20%, P <0.05). The neuroprotective effect of celastrolin Cel-H group was greater than that in Cel-L group. 3Celastrol could reduce the brain water content of ipsilateral hemispheres.Compared with Cel-L group, Cel-H group showed decline in the brain watercontent (Cel-H vs. Vehicle:82.30±0.72%vs.85.48±0.27%, P <0.05; Cel-Lvs. Vehicle:83.49±1.04%vs.85.48±0.27%, P <0.05). At Cel-L group,celastrol reduced the brain water content, but did not reach a significancelevel.4The expressions of positive cells of p-JNK, p-c-Jun and NF-κB wereobserved in ischemic cortex around infarct regions at24h post-ischemiabefore and after treatment with celastrol. Outcome of immunohistochemistryshowed that the number of positive cells of p-JNK, p-c-Jun and NF-κBdramatically increased in ischemic cortex of Vehicle group. Celastrolsignificantly reduced the positive cells of p-JNK, p-c-Jun and NF-κB afterpMCAO in Cel-H group (P <0.05). In agreement with results ofimmunohistochemistry, Western blot analysis showed that cerebralsignificantly supressed p-JNK, p-c-Jun and nuclear NF-κB levels in Cel-Hgroup (P <0.05). Simultaneously, RT-PCR analyses also showed a significantdecrease of NF-κB and c-Jun in Cel-H group (P <0.05).Conclusions: Our study showed that celastrol may have the possibility ofprotective effect against ischemic injury, and this effect may be throughdownregulation of the expression of p-JNK, p-c-Jun and NF-κB.