Andrographolide Improves MPTP-induced Parkinson Disease-like Dyskinesia And Protects Rotenone-induced Mitochondrial Dysfunction

Object: Studying the protective effect of Andrographolide on MPTP-induced Parkinson disease-like dyskinesia and rotenone-induced SH-SY5 Y cell damage to explore the role of Andrographolide on mitochondrion in Parkinson disease. Then discuss the possible protective mechanisms of Andrographolide on neuron cell to broaden the clinical indications.Methods: Using 25 mg/kg MPTP i.p. injection for 5 days to trigger Parkinson disease in mice along with the treatment of 2.5 mg/kg and 5 mg/kg Andrographolide i.p. injection for 12 days. The following motor functions of mice were analyzed by open field, pole test, rotarod, forced swimming and suspension test. To test whether Andrographolide exerts its protective impacts on neuron in midbrain, the expression of TH were determined by western blot and immunohistochemistry as well as Nissl staining. We used MDA Kit to measure the by-product of oxidative stress and SOD and Catalase Kits to determine the Reactive Oxygen Species(ROS) eliminators. For additional confirmations, TUNEL staining was used to examine the apoptosis in midbrain. Inflammation associated with peripheral and central system, as a critical evolution and progression factor in Parkinson, was investigate by ELISA and real time PCR. The activated microglia expressing Iba-1 involved in MPTP-induced mice was examine using immunohistochemistry. While the relative mitochondrial numbers and morphology were detected by transmission electron microscope, the mitochondrial copy number and copy potential were investigated by real time PCR.Different concentrations of Andrographolide incubated SH-SY5 Y cells for 3 h followed by 30 μmol/L Rotenone for 6 h in vitro. Cell viability was analyzed by MTT assay; apoptosis ratio was detected by Annexin V/PI staining; DCFH-DA dye and JC-1 dye were applied to assess ROS production and mitochondrial membrane potential respectively; ATP levels were monitored by luciferase assay; cell respiratory function was evaluated by Clark oxygen electrode. We applied MDA kit and SOD kit as it before to evaluate the levels of cellular oxidative stress. MitoTracker Greenstaining measured mitochondrial numbers through flow cytometry. Real time PCR and transmission electron microscope were applied to analyze mitochondrial copy number and evaluate the mitochondrial morphology and numbers.Results: Our results showed Andrographolide can improve the dyskinesia induced by MPTP in mice. Andrographolide enhanced the movement distance and the time spent in central area, reduced time spent on pole, increased the time on beam,improved motor coordination in water, strengthened the capture abilities in MPTP-damaged mice. What’s more, TH and protein synthesis ability in midbrain were improved in Andrographolide group. Compared with increased MDA and decreased SOD and catalase in PD mice, Andrographolide can block it and reduce the apoptosis in midbrain. The mRNA of inflammation factors in PD mice were also decreased by Andrographolide. The morphology of mitochondria were also sustained by Andrographolide. Andrographolide can prevent SH-SY5 Y cells from rotenone,complex I inhibitor, induced damage effectively. Using MTT assay, microscope and flow cytometry, Andrographolide exerted protective effect in a concentrations dependent manner. Compared rotenone treated group, Andrographolide inhibited ROS production, enhanced mitochondrial membrane potential, promoted ATP production and respiratory function to attenuate oxidative stress. Andrographolide improved not only in mitochondrial function but also in morphology. The decreased mitochondrial DNA copy number and transcription factors associated with mitochondrial copy were also improved by Andrographolide.Conclusions: The protective mechanism of Andrographolide attenuated MPTP-induced dyskinesia may be involved in anti-oxidation, anti-inflammation,maintenance of mitochondrial DNA copy number and morphology in vivo. What’s more, Andrographolide stabled mitochondrial membrane potential, inhibited ROS production, sustained mitochondrial DNA copy numbers associated with mitochondrial function along with morphology. All of these results provided foundation for the application of Andrographolide to treat Parkinson disease.