Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCδ phosphorylation

Objective The present study aims to investigate the role of protein kinase C 5 subtype （PKCS） phosphorylation in the process of 6-hydroxydopamine （6-OHDA）-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson＇ s disease. Methods The pheochromocytoma （PC 12） cell line was employed in the present study. Cells were treated with 2 μmol/L PKC5 inhibitor Rottlerin, 10 nmol/L protein kinase C α subtype （PKCα） inhibitor bisindolylmaleimide 1, or 5 nmol/L G66976 that could specifically inhibit the calcium-dependent PKC isoforms, respectively. PKC8 activator phorbol-12-myristate-13-acetate （PMA, 100 nmol/L） was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotox- icity of 6-OHDA was determined by methyl thiazolyl tetrazolium （MTT） reduction assay and PKCδ phosphorylation levels in various groups were measured by western blotting. Results Bisindolylmaleimide I and Go6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCδ phosphorylation in PC 12 cells. However, Rottlerin could inhibit the phosphorylation of PKC5 and attenuate 6-OHDA-induced cell death, and the cell viability was raised to 69.6 ±2.63% of that in control group （P 〈 0.05）. In contrast, PMA induced a significant increase in PKC5 phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to 49.8±5.06% of that in control group （P 〈 0.001）. Conclusion Rottlerin can protect PC 12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKC δ phosphorylation. The results suggest that PKCδ may be a key regulator of neuron loss in Parkinson＇s disease.

Long-term Levodopa Treatment Accelerates the Circadian Rhythm Dysfunction in a 6-hydroxydopamine Rat Model of Parkinson＇s Disease

Background： Parkinson＇s disease （PD） patients with long-term levodopa （L-DOPA） treatment are suffering from severe circadian dysfunction. However, it is hard to distinguish that the circadian disturbance in patients is due to the disease progression itself, or is affected by L-DOPA replacement therapy. This study was to investigate the role of L-DOPA on the circadian dysfunction in a rat model of PD. Methods： The rat model of PD was constructed by a bilateral striatal injection with 6-hydroxydopamine （6-OHDA）, followed by administration of saline or 25 mg/kg L-DOPA for 21 consecutive days. Rotarod test, footprint test, and open-field test were carried out to evaluate the motor function. Striatum, suprachiasmatic nucleus （SCN）, liver, and plasma were collected at 6：00, 12：00, 18：00, and 24：00. Quantitative real-time polymerase chain reaction was used to examine the expression of clock genes. Enzyme-linked immunosorbent assay was used to determine the secretion level of cortisol and melatonin. High-performance liquid chromatography was used to measure the neurotransmitters. Analysis of variance was used for data analysis. Results： L-DOPA alleviated the motor deficits induced by 6-OHDA lesions in the footprint and open-field test （P 〈 0.01, P 〈 0.001, respectively）. After L-DOPA treatment, Bmall decreased in the SCN compared with 6-OHDA group at 12：00 （P 〈 0.01） and 24：00 （P 〈 0.001 ）. In the striatum, the expression ofBmall, Rorα was lower than that in the 6-OHDA group at 18：00 （P 〈 0.05） and L-DOPA seemed to delay the peak of Per2 to 24：00. In liver, L-DOPA did not affect the rhythmicity and expression of these clock genes （P 〉 0.05）. In addition, the cortisol secretion was increased （P 〉 0.05）, but melatonin was further inhibited after L-DOPA treatment at 6：00 （P 〈 0.01）. Conclusions： In the circadian system of advanced PD rat models, circadian dysfunction is not only contributed by the degeneration of the disease itself but also long-term L-DOPA therapy may further aggravate it.