Molecular Analysis of Catecholamine Biosynthetic Enzyme Genes Induced by Mucuna pruriens

Abstract

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis of catecholamines, which are neurotransmitters involved in a wide variety of important physiological functions. The regulation of TH protein level and activity is a key step for controlling catecholamine synthesis. TH is regulated in a complex fashion by transcriptional and post-transcriptional mechanisms and is critically modulated by multiple regulatory mechanisms. TH is key molecule of the functioning of the dopaminergic system. Changes in TH expression generally reflect altered activity of catecholaminergic neurons in brain. The pathogenesis of some catecholaminergic neuron disorders, such as Parkinson's disease (PD) may be related to change in TH. In recent years, a worldwide trend towards the use of natural phytochemicals present in herbs to apply for the neurodegenerative disorders. Seeds of Mucuna pruriens (MP) have been described as a useful therapeutic agent in various diseases of the human nervous system including PD. However, at present no information is available regarding the effects of MP on catecholamine biosynthetic enzymes in animal. To explore possible neuroprotective effects of MP and related molecular mechanisms, we examined the expression of TH and AADC in rat brain. Cell viability of SH-SY5Y neuroblastoma cell line and 293 human kidney cell line was determined with different doses (0, 100, 200, 400, 800, 1000 ㎍/㎖) of MP seed treatment for 12, 24 and 48hrs. MP did not induce any cell cytotoxicity to both cell lines even with the higher doses (1000 ㎍/㎖) of MP incubated for longer time (48hrs). To determine the neuroprotective effect of MP on SH-SY5Y cells, cells were treated with rotenone or MP or MP plus rotenone, and then processed MTT assay. The result showed that the MP increased cell viability and had neuroprotective effect against rotenone. To dertermine the effects of MP on the catecholamine biosynthetic enzyme encoding genes expression, we determined changes in mRNA levels of TH and AADC, the first rate-limiting and the second enzyme in the catecholamine biosynthesis pathway. TH and AADC mRNA levels were examined in rat brain tissue by RT-PCR and real time PCR. Protein levels were determined by Western blot analysis. Rats were treated with different doses of MP (0, 100, 200, 300, and 400 ㎎/㎏) for 2, 4 and 8 hrs, respectively. The results showed that the MP significantly increased the levels of TH and AADC mRNA and protein at a dose of 400 ㎎/㎏ for 2hrs and at a dose of 200㎎/㎏ for 4hrs. However, mRNA and protein levels of both genes were not change at different doses for 8h. The mRNA and protein levels of AADC were induced by MP at different doses and times. These results indicate that MP has a complex and differential effect on TH and AADC gene expression and it depends on the treatment dose and duration of administration. In conclusion, MP did not show any cell cytotoxicity to human neuroblastoma SH-SY5Y and human kidney 293 cell lines and had the neuroprotective effects against rotenone. Futhermore, both catecholamine biosynthetic enzymes encoding genes (TH and AADC) were up regulated by the treatment of MP. Therefore, it could have therapeutic value in the treatment and improvement of PD.