Neuroprotective Effect Of Oleracein E On Parkinson’s Disease Models

Parkinson’s disease (PD) is the second common age-related neurodegenerative disease. It is pathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the decrease of dopamine content in the striatum. It is often accompanied by motor symptoms, such as tremor, rigidity, bradykinesia and gait disorders, which weaken the motor ability of the patient and influence patients’ normal life. Currently, levodopa is the most common clinical drug for the treatment of PD. It can compensate for the dopamine deficiency and alleviate the symptom of disease, however, it cann’t protect the neuron cells. Long-term usage of levodopa even cause some side-effects on patient. Therefore, it is still urgent to develop the drug for the treatment of PD.Portulaca oleracea L. (purslane) is used as a vegetable and also a medicinal drug in China. It has many kinds of functions such as heating-clearing, detoxifying, dispelling heat from blood to stop bleeding. The pharmacological study showed that P. oleracea possessed a wide range of activities, including antioxidant, anti-dementia, analgesia, anti-anxiety and neuroprotective functions. Most recently, it was found that the extract of P. oleracea could improve the motor skill in neurotoxins (such as rotenone) induced PD animal models, protect neuron cells, and increase the dopamine content in striatum. However, to date, the active constituents responsible for the anti-PD function of P. oleracea is not known yet.Oleradein E (OE) is a tetrahydroisoquinoline alkaloid isolated from P. oleracea. In viewing of the fact that OE possessed potent antioxidant activity and alleviated oxidative stress, ameliorated hippocampal neuronal damage and apoptosis, and elevated spatial memory capacity in senescent mice induced by large dosage of D-galactose and NaNO2, we hypothesized that OE is one active compound for the anti-PD function of P. oleracea. In this paper, we evaluated the effects of OE on PD cellular model and animal model.(1) The PD cellular model was established by exposing SH-SY5Y neuroblastoma cell to 5 μM rotenone for 24 h. LDH assay, staining of TUNEL, DCFH-DA and JC-1 were used to measure the cell viability, apoptosis ratio, ROS level and mitochondrial membrane potential, respectively. Western blot is used to determine the protein expression. The result showed that treatment of rotenone for 24 h significantly increased the LDH release rate (p<0.001), apoptosis ratio (p<0.001) and ROS level (p<0.001), while evidently decreased the mitochondrial membrane potential, in comparison with the vehicle control. Treatment of rotenone for 1 h,12 h, and 24 h didn’t influence or slightly decreased t-ERK expression, however, it evidently increased expression of p-MEK and p-ERK, when compared with vehicle control. In particular, ERK 1/2 phosphorylation in rotenone toxified cells significantly increased at 24 h (p<0.001). Moreover, rotenone upregulated the protein expression of bax/bcl-2 (p=0.005), cytochrome c (p<0.001) and cleaved caspase-3 (p<0.001). In comparison with rotenone model, pretreatment with OE (10 μM) for 2 h and cotreatment with OE for 24 h significantly decreased LDH release rate (p<0.001), apoptosis ratio (p<0.001) and ROS level (p<0.001) in SH-SY5Y cells, and upregulated the mitochondrial membrane potential. OE evidently downregulated the expression of p-MEK and p-ERK after treatment of rotenone for 1 h,12 h and 24 h, and inhibited against ERK phosphorylation reached to the significant level at 24h (p= 0.002). Furthermore, OE significantly decreased the expression ratio of bax/bcl-2 (p= 0.009), and inhibited the expression of cytochrome c (p=0.012) and cleaved caspase-3 (p=0.003).(2) The PD C57BL-6J mouse model was established by oral administration of rotenone (30 mg/kg/d) for 56 days. Selegiline hydrochloride (positive control,10 mg/kg/d), low dose of OE (3 mg/kg/d) and high dose of OE (15 mg/kg/d) were orally administrated after treatment of rotenone for 1 h. Spontaneous activity test, rota-rod test and gait test were performed to evaluate the motor ability of mouse. SOD activity and MDA content in the brain and plasma were measured to assess oxidative stress level. Western blot assay was used to detect expression of p-ERK and apoptosis-related proteins in the midbrain and striatum of mouse. Results showed that after treatment of mice with rotenone for 56 days, the distance moved in the spontaneous activity test (p=0.028), the time sustained on the rod (p=0.033), the coordination index (p<0.001) and stride length (p<0.001) of mouse significantly decreased and the stride width (p=0.01) significantly increased. SOD activity in the striatum (p=0.031) and blood plasma (p<0.001) of PD mouse also decreased while MDA content increased in the midbrain (p=0.005), striatum (p=0.045) and blood plasma (p<0.001)of PD mouse. The TH positive cell numbers (p=0.034) and the density of TH positive fibers (p<0.001) in the SNpc significantly decreased, and the axons and dendrites of neuron became shorter. Besides, p-ERK 1/2 expression significantly increased in the midbrain (p=0.021)and striatum (p<0.001) of PD mice. Compared with rotenone model, high dose of OE improved moving distance (p = 0.001), sustained time on the rod (p=0.024), coordination index (p=0.011) and stride length (p=0.048). In addition, it increased SOD activity (p=0.007,P=0.022), decreased MDA content (p=0.005, p=0.015) and reduced ERK phosphorylation (p =0.043, p=0.037), in the midbrain and striatum of PD mice. Furthermore, it alleviated the dopaminergic neuronal injury, increased the TH positive cell numbers (p=0.004) and the density of TH positive fibers (p=0.001). Low dose OE could only significantly increase coordination index (p=0.002), stride length (p=0.016), TH positive cell numbers (p=0.025) and the density of TH positive fibers (p=0.012), and decrease stride width (p=0.005), however, it had no effects on other parameters. Compared with rotenone model, selegiline hydrochloride increased the time sustained on the rota-rad (p=0.029), coordination index (p=0.025), stride length (p=0.029), SOD activity in the plasma (p=0.002) and the density of TH positive fibers (p= 0.009), while decreased the stride width (p=0.004), MDA content (p<0.001, p= 0.005, p<0.001) in the midbrain, striatum and plasma of mouse. Above results indicated that, similar with the function of selegiline hydrochloride, OE can improve the motor ability of PD mouse model, through alleviating oxidative stress and inhibiting dopaminergic neuronal damage. Whereas, different from selegiline hydrochloride, OE can inhibit phosphorylation of ERK1/2.