DNA的損傷和修復(fù)對多巴胺能神經(jīng)元存亡的作用及其機制.pdf

1、The pathogenesis of Parkinson's disease (PD) remains obscure, but there is increasing evidence that oxidative damage to DNA, especially mitochondrial DNA, may play an important role in dopaminergic neurodegeneration in t

2、he substantia nigra pars compacta (SNC) of patients with PD. Therefore, the supplementation with antioxidants or improvement of DNA repair activity,which prevent or reduce the excessive accumulation of DNA oxidative dama

3、ge,may hold particular promise as powerful neuroprotective therapies in the treatment of PD. In the present study, we evaluate the effect of melatonin, a potentfreeradicalscavengerandantioxidant,onMPTP/MPP+induced mitoch

4、ondrial DNA oxidative damage in vivo and in vitro. Also, the role of excision repair cross complementing 1(ERCC1) overexpression, which is an important nucleotides excision repair factor, on MPP+-induced DNA oxidative da

5、mage is investigated. Results showed as follows:Part Ⅰ: Melatonin Protect against MPTP/MPP+ -induced Mitochondrial DNAOxidative Damage in vivo and in vitro The effects of melatonin on the mitochondrial DNA (mtDNA) damage

6、 induced by1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine(MPTP)and 1-methyl-4-phenylpyridine ion (MPP<'+>) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivit

7、y of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the SNC of mouse brain. Melatonin preinjection (15or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA

8、oxidative damage.In SH-SY5Y cells, MPP<'+> (1 mM) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment.Melatonin (200μM) preincubation could significantly attenu

9、ate MPP<'+>-induced mitochondrial DNA oxidative damage. Furthermore, MPP<'+> time-dependently increased the accumulation of mitochondrial oxygen free radicals (OFR) from 1to 24 hr and gradually decreased the mitochondria

10、l membrane potential (Ψm)from 18 to 36 hr after incubation. At 72 hr after incubation, MPP<'+> caused about 49 ﹪ cell death. However, melatonin could prevent MPP+-induced OFR generation and Ψm collapse, and later cell de

11、ath. The present results suggest that cytoprotectionof melatoninagainst MPTP/MPP<'+>-induced celldeath may associate with the attenuation of mitochondrial DNA oxidative damage via inhibiting mitochondrial OFR generationa

12、nd the prevention of Ψm collapse Part Ⅱ: Overexpression of ERCC1 protects against MPP<'+>-induce oxidativeDNA damage in vitroInthepresentstudy,theroleofERCC1overexpressionon1-methyl-4-phenylpyridinium (MPP<'+>)-induce ox

13、idative DNA damage and cellapoptosis was investigated in PC12 cells. Oxidative DNA damage and theexpression of ERCC1 protein were determined by immunostaining.The resultsshowed that administration of MPP+ in the culture

14、medium at the finalconcentration of 1 mM could time-dependently elevate the amounts of ERCC1expression and 8-hydroxyguanine (8-oxoG), a commonest oxidized base, in thecells. Furthermore, we established PC12 cell lines st

15、ably expressing humanERCC1 to investigate the role of ERCC1 overproduction in MPP<'+>-inducedcytotoxicity. Oxidative DNA damage was time-dependently increased in controlcells from 6 to 48 hr after MPP+ exposure (p<0.05 v

16、ersus vehicle). However, inthe PC12 cells overexpressing ERCC1, MPP<'+>-induced oxidative DNA damagewas delayed and significantly decreased compared with the control cells.Besides, overexpression of ERCC1 also attenuated

17、 apoptosis and increased cellsurvival in the cultures at 48 htafter MPP+ treatment. The results suggest thatthe protection of ERCC1 overexpression against MPP<'+>-induced PC12 cell injuryis related, at least partly, to i

18、ts ability to attenuate DNA oxidative damage.Keywords:MPP+;ERCC1;overexpression;DNAoxidativedamage; 1-methyl-4-phenylpyridinium; 8-hydroxyguaninePart Ⅲ: ConclusionOxidative damage to DNA, especially mitochondrial DNA, ma