Protective Effects And Mechanisms Of Rosmarinic Acid Against Rotenone-induced Parkinson’s Disease Models

Parkinson’s disease(PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra(SN) and the appearance of fibrillar aggregates of alpha-synuclein called Lewy bodies(LBs), with clinical symptoms of bradykinesia, rigidity, resting tremor and postural instability. Missense mutations in alpha-synuclein gene(e.g. A53 T, A30 P, E46K) found in familial PD patients were reported to be implicated in the pathogenesis of PD. Studies have demonstrated alpha-synuclein aggregation is the key factor in the pathogenesis of PD. Research in the molecular or drugs which can reduce alpha-synuclein has become an important strategy for the treatment of PD.Rosmarinic acid(RA) is an ester of caffeic acid and 3, 4-dihydroxyphenyllactic acid, which is commonly found in various plant families. RA has a number of interesting biological activities, e.g. antiviral, antibacterial, anti-inflammatory and antioxidant activities. Our previous studies have confirmed that RA had a neurorescue effect on nigral dopamine neurons in 6-hydroxydopamine(6-OHDA)-induced rat model of PD. The mechanism may be related to the antioxidative effect and reduction of iron level in SN. Furthermore, RA could protect against 1-methyl-4-phenylpyridium(MPP+) and 6-OHDA induced neurotoxicities through its antioxidative property in vitro. Previous studies indicated that RA might play an important role in the prevention and treatment of PD. However, the molecular mechanisms underlying this neuroprotective effect are still not elucidated. As alpha-synuclein missense mutations and abnormal aggregation could lead to dopaminergic neurons degeneration, and oxidative stress could lead to alpha-synuclein abnormal aggregation, we hypothsis that RA might inhibit alpha-synuclein abnormal aggregation through its antioxidative effects. It has been demonstrated that RA could inhibit alpha-synuclein fibers formation in vitro. However, weather the protective effect of RA is related to the inhibition of alpha-synuclein aggregation and the possible mechanisms involved in this process are still unclear.Rotenone, which is commonly used as a liposoluble insecticide, is widely believed to be a high-affinity, specific inhibitor of mitochondrial complex I. It is widely used in copying the pathological changes of PD by inducing energy repletion of dopaminergic neurons in the SN. Rotenone-induced PD model seems to replicate almost all of the hallmarks of PD including alpha-synuclein aggregation, LBs formation and nigral dopaminergic neurons loss. Therefore, in this study, immunofluorescence, real time PCR, western blots, flow cytometry and other methods were applied to investigate the effects of RA against rotenone-induced alpha-synuclein aggregation and to explore the possible mechanisms involved in this process. The results were shown as follows:1. The residence time of mice which had orally received rotenone for 56 d on rotating treadmills was decreased by 47%, compared with control(P<0.05); After pretreatment with 20 or 50 mg/kg RA, the residence time on rotating treadmills was increased by 83% or 79%, compared with rotenone group(P<0.05). 2. The number of TH-positive neurons and the expression of TH were decreased by 42% and 28% in the SN of rotenone-treated mice for 56 d, compared with control(P<0.05); After pretreatment with 20 or 50 mg/kg RA, the number of TH-positive neurons was increased by 28% or 41%, and TH expression was increased by 15% or 30%, compared with rotenone group(P<0.05). 3. The expression of alpha-synuclein was increased by 37% in the SN of rotenone-treated mice for 56 d, compared with control(P<0.01); After pretreatment with 20 or 50 mg/kg RA, alpha-synuclein expression was decreased by 19% or 22%, compared with rotenone group(P<0.05). 4. Cell viability was decreased by 33% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.001); After pretreatment with 10-5 M~10-10 mol/L RA, cell viability was increased by 18%, 21%, 14%, 10%, 18% or 14%, compared with rotenone group(P<0.05). 5. The mitochondrial transmembrane potential(ΔΨm) was decreased by 10% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.001). The expression of Bcl-2 or p-Bad was decreased by 56% or 40%, compared with control(P<0.05). After pretreatment with 10-9 or 10-5 mol/L RA, ΔΨm was increased by 8% or 6%, compared with rotenone group(P<0.001). Bcl-2 expression was increased by 61% or 76%; p-Bad expression was increased by 46% or 51%, compared with rotenone group(P<0.05). 6. The m RNA expression of alpha-synuclein was increased by 344% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.01); After pretreatment with 10-9 or 10-5 mol/L RA, alpha-synuclein m RNA expression wasdecreased by 66% or 52%, compared with rotenone group(P<0.05). A-synuclein aggregation was observed in the rotenone group. Almost none α-synuclein aggregation could be observed in RA pretreatment groups. 7. After treatment with 500 nmol/L rotenone for 24 h, reactive oxide species(ROS) generation was increased by 139% and SOD expression was decreased by 42% in SH-SY5 Y cells, compared with control(P<0.001); After pretreatment with 10-9 or 10-5 mol/L RA, ROS generation was decreased by 57% or 75%, compared with rotenone group(P<0.001). SOD expression was increased by 35% or 32%, compared with rotenone group(P<0.01). 8. The expression of heme oxygenase 1(HO-1) was decreased by 50% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.001); After pretreatment with 10-9 or 10-5 mol/L RA, HO-1 expression was increased by 52% or 37%, compared with rotenone group(P<0.05). 9. The expression of NF2E2-related factor 2(Nrf2) was increased by 71% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.01); After pretreatment with 10-9 or 10-5 mol/L RA, Nrf2 expression was increased by 25% or 40%, compared with rotenone group(P<0.05). 10. The expression of hypoxia-inducible factor-1α(HIF-1α) or hypoxia-inducible factor-2α(HIF-2α) was decreased by 76% or 37% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.01); After pretreatment with 10-9 RA, HIF-1α or HIF-2α expression was increased by 39% or 43%, compared with rotenone group(P<0.05). 11. The expression of heat shock protein 70(HSP70) was decreased by 44% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.001); After pretreatment with 10-9 or 10-5 mol/L RA, HSP70 expression was increased by 33% or 33%, compared with rotenone group(P<0.01). 12. The phosphorylation level of extracellular signal regulated kinases(ERKs) was decreased by 77% in SH-SY5 Y cells treated with 500 nmol/L rotenone for 24 h, compared with control(P<0.001); After pretreatment with 10-9 or 10-5 mol/L RA, phosphor-ERK1/2 level was increased by 75% or 100%, compared with rotenone group(P<0.01). MEK inhibitor PD98059(10 μM) pre-incubation for 0.5 h could fully abolish RA induced up-regulation of HO-1 or HSP70. HO-1 or HSP70 expression was decreased by 30% or 25%, compared with 10-9 mol/L RA/rotenone group(P<0.05).The above results suggest that RA could protect dopaminergic neurons through inhibiting alpha-synuclein up-regulation in PD. The mechanisms may be related to its antioxidant effect by up-regulating the expression of SOD and promote the degradation of alpha-synuclein by up-regulating the expression of HO-1 and HSP70. The up-regulation of the HO-1 may be due to Nrf2 and HIF-1α. Intracellular signaling pathway MEK/ERK might participate in these processes. Moreover, these results provide new findings and new strategies for the prevention and treatment of PD.