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Ginsenoside Rd Protects Against Cerebral Ischemic Stroκe in Rats by Affecting Poly(ADP-ribose)

Author: HuGengYao
Tutor: ZhaoGang; ShiMing
School: Fourth Military Medical University
Course: Neurology
Keywords: Ginsenoside Rd Poly(ADP-ribose) polymerase NF-κB DNA repair Cerebral ischemia
CLC: R285.5
Type: Master's thesis
Year: 2012
Downloads: 49
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Abstract


Few diseases are as destructive and complex as stroke, which has beenbecoming the second cause of death threaten our survival and health. However,drugs for treatment of stroke clinically are not well successful. So there iscompelling need to accelerate efforts to explore new avenues to conquer stroke.Gingseng has been considered as a typical tonic by the fast east countriesduring the past2,000years, and this rare Chinese herb medicine is well enjoyedby people all over the world now. Ginsenosides(GS) are extracted fromrootstock of five plus seccoplant Gingseng, and there are40different types ofginsenosides. Abundant researches on ginsenoside suggest GSRd is helpful inneuron nutrition and neuroprotection aspects, can resist atherosclerosis, enhancelearning and memory, delay aging, and so on. At present, ginsenoside isbecoming a hot point among researches due to numerous medical value. GSRd is a monomer of wide biological activities. Our previous studies clinically andbasically indicate GSRd play a vital role in neuroprotection after ischemia.Researches in rats show GSRd can resist glutamate excitotocity injurythrough inhibiting Ca2+influx; can also relieve oxidative damage andinflammatory lesions; can ease mitochondrial dysfunction and apoptosis processafter focal cerebral ischemia in rats. All above indicate GSRd has obviousneuroprotection effect and may become a new candidate drug for cerebralischemic stroke. However, it’s not clear how GSRd comes into play, so it’simportant to elaborate the mechanism for clinical application of GSRd. Ourlatest study shows GSRd can decrease apoptosis inducing factor(AIF) level aftercerebral ischemia in rats, and Poly(ADP-ribose)(PAR) can cause AIF totranslocate from mitochondria to nucleus and induce cell death.PAR is produced by PARP[Poly(ADP-ribose) polymerase,PARP] familymembers, the latter are abundant proteins combined with nuclear matrix proteinin mammals, and PARP-1is the most abundant and extensive functionary of17related family members. PARP-1is the first protein to be found to havePARylation activity, which can use NAD+as a donor of ADP-ribose units ofstraight or branching, and catalyzes the covalent attachment of PAR polymers onitself and other nuclear related proteins. Then PARG degrade the PAR unitsfrom polymers and keep its normal level and circulation in body. Thepost-translational modifications as PARylation on protein targets, has closelyrelated to the chromosomes of the stability, DNA repair, gene transcription, andso on. Under normal conditions, PAR has a low level in cells and few PARlinked with proteins; but under pathological conditions including cerebralischemia, activation of PARP-1can lead to PAR polymers promptly. There aretwo different aspects of PARP-1activation effects:(1) PAR product generated by PARP-1activation can act on nuclear related protein targets, such as DNArepair protein, histone protein, and promote DNA repair.(2) PAR translocated tonucleus and promote AIF assembling in nucleus from mitochondria and thusaggravate DNA injury and cell death; PARP-1activation can lead to NF-κBaccumulation in nucleus and promote NF-κB related gene expression, which isinvolved in inflammatory respond. So, PARP-1activation mildly benefits cellrepair under slight stimuli, but overactivation of PARP-1can induce NF-κBsignal pathway and AIF translocated to nucleus to cause cell death.Our latest research demonstrates that GSRd can decrease AIF level aftercerebral ischemia in rats, which indicate GSRd can exert neuroprotectioneffectthrough affecting AIF related PARP-1pathway. This research is mainly todiscuss the relationship between GSRd and PAR polymer by using MCAOmodel in rats, elucidate futher mechanism of neuroprotection effect of GSRdafter acute cerebral ischemia.Experiment1The effect of GSRd on PAR polymer in the superacute stageafter MCAO in ratsObjective: To investigate the effect of GSRd pretreatment on PARpolymer expression in rats in the superacute stage after focal cerebralischemia(2h). Methods: A total of60male SD rats weighing280-300g wererandomly divided into sham operation group (Sham group, n=20),propanediolpretreatment group (Vehicle group, n=20) and GSRd pretreatment group (Rdgroup, n=20). The transient focal ischemia/reperfusion was induced byintervening a MCAO monofilament from the right external carotid artery into the origin of the middle cerebral artery and removing it after2h. In Vehiclegroup and Rd group, rats were respectively exposed to propanediol andGSRd(10mg/kg)30min before the occlusion of the right middle cerebralartery. The rats in Sham group were operated the same as the propanediolgroup, but cerebral artery was not blocked. The expression of PAR polymerwas detected by westernblotting and immunohistochemistry at2h afterMCAO in rats. Results: Our results showed that PAR level in Vehicle groupand Rd group were increased compared with the ones in the Sham group (P<0.01); PAR level in Rd group was significantly improved compared withVehicle group (P<0.01). Conclusion: The results suggest that GSRd (10mg/kg) can obviously increase the expression of PAR polymer in thesuperacute stage after MCAO.Experiment2The effect of GSRd on PAR polymers and NF-κB in the acutestage after MCAO in ratsObjective: To investigate the effect of GSRd pretreatment on PAR polymer、NF-κB in rats after focal cerebral ischemia(12h-24h). Methods: Rats underwentmiddle cerebral artery occlusion, and PAR polymer and NF-κB were detected at12h、18h、24h after ischemia. SD rats was randomly divided into Normal group、GSRd alone group、MCAO group、MCAO+Rd group. Western blotting was usedto determine PAR polymer and NF-κB levels after middle cerebral arteryocclusion at12h、18h、24h, respectively. Results:Western blotting detectionindicated that PAR polymer and NF-κB level in nucleus obviously increasedafter cerebral ischemia injury (12h-24h); then their level fall sharply after10 mg/Kg GSRd pretreatment before MCAO. Conclusion:GSRd can lower PARpolymer and NF-κB in nucleus after ischemia, and thus resist cerebralischemia/reperfusion stress. It indicates that GSRd can exert its neuroprotectioneffect through PARP-1pathway after cerebral ischemia.Experiment3Research on GSRd acting directly on PARP activityObjective: To verify whether GSRd can act directly on PARP and inhibit it’sactivity, then take neuroprotection effect after cerebral ischemic stroke.Methods: HT Universal Chemiluminescent PARP Assay Κit with Histone-coatdStrip Wells is mainly used to sift possible inhibitor of PARP and IC50of theinhibitor, all the operating process follows the instruction. The specific group isas follows:1) PARP group;2) PARP+1μM Rd group;3) PARP+10μM Rd group;4) PARP+100μM Rd group;5) PARP+100μM3-AB group. Results:Comparedwith control group, absorbency of100μM3-AB group was obviouslydecreased,which indicated3-AB can inhibit PARP activity. PARP+Rd groups(1μM、10μM、100μM Rd) have no obvious difference in absorbency comparewith control group. Conclusion:GSRd can not affect absorbency of PAR, whichindicates GSRd can not inhibit PARP activity directly.

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