| Objective:Parkinson Disease(PD) is a neurodegenerative disease with multifactorial etiopathogenesis. Theacrine(1,3,7,9-tetramethyluric acid), whose chemical structure is similiar with purine alkaloids, caffeine and theophylline, is suggested to have neuroprotective effects on Parkinson disease or other neurodegenerative disease according to our previous studies. In this study, we investigated the protective effects of Theacrine on dopaminergic nerons and its related mechanisms in different in vivo and in vitro PD models. Methods:SD rats with Parkinson disease were randomly divided into 4 groups: control group, 6-OHDA group, Theacrine-L(6-OHDA + 10 mg/kg Theacrine), Theacrine-H(6-OHDA + 20 mg/kg Theacrine) and positive selegiline group(6-OHDA + 10 mg/kg selegiline). In drug treatment groups, rats were administrated of Theacrine and selegiline for 2 weeks. The control group and 6-OHDA group received water only. To evaluate the protective effects of Theacrine on SD rat model of Parkinson's disease induced by the 6-OHDA, the behavior tests including rotation test, rotarod test and CatWalk test were conducted. Immunohistochemical examination was also conducted to detect the TH-positive neuron numbers in the Substantia Ingra. In addition, the levels of dopamine(DA) and its metabolites contents dihydroxyphenyl acetic acid(DOPAC) and homovanillic acid(HVA) in striatum were determined by high performance liquid chromatography with an electrochemical detection(HPLC-ECD).Zebrafish embryos were treated with MPTP(40 ?g/ml) and Theacrine(40 1 mM) together from 1 dpf to 3 dpf. Depranyl(20 ?g/ml)was used as positive controls. DA neurons were detected with whole mount in situ hybridization with anti-sense dat probe. Behavior was measured with tactile response assay. The result showed that Theacrine rescued the DA neuron loss and abnormal behavior induced by MPTP.The in vitro experiment was performed to evaluate the neuroprotective of Theacrine on MPP+-caused SH-SY5 Y cell damage model. Cells were randomly divided into 3 groups, including Control, MPP+, and “MPP+ + Theacrine”. In the “MPP+ + Theacrine” group, cells were treated with 200 ?M of Theacrine and cultured for 24 h. Then all cells except control ones were treated with MPP+(2 mM). Cells were harvested for LDH and SOD activitytest. ROS generation rate was detected by fluorescent probe, mitochondrial membrane potential and cell apoptosis analysis by flow cytometry measurment.Another experiment was conducted to explore the related mechanisms of Theacrine on 6-OHDA-established rat PD. The substantia nigra were harvested to examine the protein expressions of Sirt3, p-Akt/Akt ? Bcl2/Bax ? Clealved Caspase3/Caspase3 and Cyt-c and. The effects of Theacrine on Sirt3 activation, the activities of complex II and V, and the acetylation of SOD2 were detected respectively by Sirt3 direct fluorescent screening assay kit, Complex II and V Immunocapture Kit and immunoprecipitation. Results:In the 6-OHDA induced rat PD model, Theacrine displayed a significant neuroprotective effects, reflected by decreased rotation behavior induced by apomorphine, increased latency on the rod, and improved print view and footfall patterns including the stands time, step cycle and swing speed in the catwalk behavior test. The immunohistochemical results showed that Theacrine could significantly increase protein expression of tyrosine hydroxylase(TH) on the impaired side. The results showed that Theacine rescueed the loss of DA neurons induced by MPTP. It was also found to improve the behaviors in Zebrafish PD model.CCK-8 and LDH assay showed thatTheacrine plays a protective effect on MPP+-caused SH-SY5 Y cell damage. In the meantime, Theacrine significantly limited oxidative damage by inhibiting excessive ROS generation and improve SOD activity, and maintained mitochondrial membrane potential. PI/Annexin V staining combined with flow cytometry showed that Theacrine significantly drecreased cell apoptosis ratio in MPP+-treated SH-SY5 Y. Moreover, Theacrine significantly decreased the expression of the SOD2 acetylation level, elevated mitochondrial respiratory chain Complex II and V activities. Further studies revealed small interfere RNA(siRNA) partially nullified the protective effects induced by MPP+. Knockdown of Sirt3 by siRNA interference, cleaved Caspase3 increased significantly. On the contrary, overexpression using Sirt3 targeted plasmid, the expression of apoptosis protein Cleaved Caspase3 was significantly lowered. Besides, an obvious increase in protein expression of p–Akt, Bcl-2/Bax rate and related apoptosis pathway protein expression were also found in Theacrine-treated group. Conclusion:Theacrine showed potential protecvtive effects on dopaminergicneuronsin 6-OHDA rat PD model, Zebrafish PD model, and MPP+ cell model. The mechanism was related with the activation of Sirt3, through which to elevate the activities of the mitochondrial protein such as SOD2, complex II and V, thus decreasing ROS generation and increase ROS clearence to inhibit apoptosis. |
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