Neuroprotective Roles And Mechanisms Of Rapamycin In6-hydroxydopamine-induced Cell And Rat Models Of Parkinson’s Disease

ObjectiveParkinson’s disease (PD) is a neurodegenerative disease of central nervous system,in which oxidative stress and neuronal apoptosis play a key role in its pathogenesis.Rapamycin, a widely used immunosuppressant, is involved in some pathophysiologicalprocesses by inactivating the target of rapamycin (TOR). In a series ofneurodegenerative diseases, rapamycin displayed crucial significance in theirtherapeutic pathogenesis. However, role of rapamycin in PD therapy is not well known.In view of this, this study pre-treated6-hydroxydopamine (6-OHDA)-induced cell andrat models of PD with rapamycin to explore its roles and mechanisms inneuroprotections.MethodsIn vivo, for the induction of PD,6-OHDA (4μg/μl,2μl/site) was injected into2different sites within the right striatum of female Sprague-Dawley (SD) rats. SD ratswere randomly divided into the following5groups: Sham-operated (group S); PDmodel (group P); Pre-treated with a low (group L-R,0.05mg/kg/d), moderate (groupM-R,0.5mg/kg/d) or high (group H-R,5mg/kg/d) dose of rapamycin. Different doses ofrapamycin or vehicle were intragastrically administered once daily to the rats from day7before the induction of PD. On day25all animals underwent rotational testing byusing apomorphine (0.5mg/kg) to evaluate the motor asymmetry caused by the unilateral nigrostriatal lesion. On day28, the rats were sacrificed by cardiac perfusionand the brains were removed. Tyrosine hydroxylase (TH)++neurons in the substantianigra were evaluated by immunohistochemistry. Neuronal and mitochondrialultrastructures were detected by transmission electron microscope (TEM). Peroxidelevels and antioxidant activities in the midbrain were measured by assay kits, includingmalondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX). After total RNA was extracted, semi-quantitative RT-PCR was performedto examine the mRNA levels of Bcl-2, Bax and P47phox. The protein expressions ofBcl-2, Bax, cytochrome c and cleaved caspase-9were detected by western blot.In vitro, cell PD model was induced by various concentrations of6-OHDA for24hin PC12cells. The damaged PC12cells induced by6-OHDA were pre-treated withvarious concentrations of rapamycin at different time points. The viability of damagedPC12cells was determined by cell counting kit-8(CCK-8). After selection of optimalconcentration, the damaged PC12cells induced by6-OHDA were pre-treated with3μMrapamycin for6h. The cycles of damaged PC12cells were analyzed by propidiumiodide (PI) staining kit in a flow cytometer, which was also employed to detect theapoptosis of PC12cells by Annexin Ⅴ-FITC and PI double staining kit. Afterrapamycin-pretreated PC12cells were induced by6-OHDA for1h, reactive oxygenspecies (ROS) were measured by an assay kit containing a DCFH-DA probe. Followingextraction of total RNA in treated PC12cells, real-time reversetranscriptional-polymerase chain reaction (RT-PCR) was performed to determine geneexpressions of P47phox, TH, hemo oxygenase-1(HO-1) and transcriptional factorNF-E2-related factor2(Nrf2).ResultsIn vivo experiment:(1) Pre-treatment with various doses of rapamycin significantlyreduced the number of contralateral rotations as compared with those in group P(p<0.05).(2) The number of remaining TH+neurons in the substantia nigra from groupP rats was significantly lower as compared with that from group S rats (p<0.001). Pre-treatment with rapamycin significantly increased the number of TH+neurons in thesubstantia nigra as compared to group P (p<0.001).(3) TEM detections demonstratedcondensed chromatin in shrunken nuclei in the neurons of PD model rats. Themitochondria were evidently swelled and vacuolated, and the cristae were lessened,distorted or even disappeared. However, in the rats pre-treated with rapamycin，ultrastructual injuries were significantly alleviated.(4) In group P rats, MDA levelswere elevated but SOD and GSH-PX activities were decreased as compared with thosein group S rats (p<0.001). Following pre-treatment with rapamycin, a reduction in MDAlevels and an increase in SOD and GSH-PX activities were observed as compared withthose in group P (p<0.05).(5) In group P rats, RT-PCR analyses showed lower Bcl-2but higher Bax mRNA levels than those in group S rats (p<0.01). Pre-treatment withrapamycin significantly increased Bcl-2and decreased Bax mRNA levels as comparedto pre-treatment with the vehicle (group P)(p<0.01). Western blot analyses revealedsimilar results to RT-PCR detection in protein expression of Bcl-2and Bax (p<0.05).(6)In group P rats, RT-PCR analyses showed that the P47phoxmRNA levels weresignificant elevated as compared with those in group S rats (p<0.001). Pre-treatmentwith rapamycin significantly reduced P47phoxmRNA expression as compared topre-treatment with vehicle (group P)(p<0.01). Western blot analyses demonstrated thatthe release of cytochrome c and the expression of cleaved caspase-9were increased ingroup P rats, as compared with those in group S rats (p<0.001). Pre-treatment withrapamycin significantly suppressed cytochrome c release and caspase-9expression ascompared with pre-treatment with the vehicle (group P)(p<0.05). In addition, all theresults including behavioral rotations, remaining TH+neurons, oxidative stressindicators, as well as expressions of Bcl-2, Bax, P47phox, cytochrome c and cleavedcaspase-9showed no statistical differences among all doses of rapamycin pre-treatedgroups.In vitro experiment:(1) PD cell models were successfully induced by100μMconcentration of6-OHDA for24h in PC12cells, which resulted in57.67±9.84%of cellviability.(2) As compared with blank control, mono-treatment with0.5μM-15μM rapamycin for24h exhibited no impact on the viability of normal PC12cells (p>0.05).However, mono-treatment with rapamycin for48h and72h showed inhibition of theviability of PC12cells.(3) Pre-treatment with0.5μM-3μM rapamycin for3h-12hsignificantly elevated the viability of damaged PC12cells induced by6-OHDA(p<0.05), of which pre-treatment with3μM rapamycin for6h offered an optimalcondition (p<0.01).(4) Cycle analyses demonstrated that6-OHDA remarkablyincreased the percentage of PC12cells in G0/G1phase (p<0.001), and decreased thepercentage of PC12cells in S and G2/M phase (p<0.01). Following pre-treatment with3μM rapamycin for6h, in damaged PC12cells, the percentage of G0/G1phase wasobviously reduced (p=0.018) and the percentage of S phase was elevated (p=0.028) ascompared with6-OHDA group.(5) Mono-treatment with rapamycin had no effect onapoptosis of normal PC12cells. However,6-OHDA induction significantly promotedapoptosis of PC12cells (p<0.001), which could be remarkably alleviated bypre-treatment with3μM rapamycin for6h (p<0.05).(6) There was no differencebetween normal and rapamycin mono-treated cells on ROS levels, and6-OHDAsignificantly promoted the production of ROS in damaged PC12cells (p<0.01).However, pre-treatment of rapamycin resulted in a reduction of ROS levels in6-OHDA-induced PC12cells (p<0.05).(7) After mono-treatment with3μM rapamycinfor6h-12h, mRNA expressions of HO-1and Nrf2were higher than in normal PC12cells (p<0.05), which fell to baseline levels in24h-48h.(8)6-OHDA inductionobviously increase P47phoxbut reduced TH mRNA expressions in PC12cells (p<0.05),which were reversed by pre-treatment with3μM rapamycin for6h (p<0.05). HO-1andNrf2mRNA expressions in damaged PC12cells induced by6-OHDA were higher thanthose in normal PC12cells (p<0.05), which were further up-regulated by pre-treatmentwith rapamycin (p<0.05).ConclusionsRapamycin could provide behavioral improvements, protect against the loss ofdopaminergic neurons and reduce expressions of pro-apoptotic markers in PD rats, which was consistent with enhanced viability and reduced apoptosis of damaged PC12cells in rapamycin-pretreated PD cell models. In PD, enhanced capabilities againstoxidative stress and changes in cell cycles may play a key role in the neuroprotectivemechanisms of rapamycin. In view of this, recovery of equilibrium between pro-andanti-oxidative stress signals may provide a promising strategy in PD therapy.