Neuroprotective Effects of Detoxified and Non-detoxified Rhus verniciflua Stokes: Regulation of Catecholamine Biosynthesis and Neurotrophic Factors

Abstract

Parkinson’s disease (PD) is a multifactorial chronic progressive neurodegenerative disorder after Alzheimer’s disease (AD) and is one of the major causes of morbidity in the modern society, which impairs quality of life. PD is caused by the loss of dopamine neurons of the substantia nigra pars compacta (SNc) with a concomitant loss of dopamine (DA) in the striatum. Although the underlying mechanism for selective degeneration of dopaminergic (DArgic) neurons are unknown, oxidative stress, apoptosis, mitochondrial dysfunction, and proteasomal dysfunction are suggested as contributing factors. Among the factors contributing to the burdensome cost of managing PD is the fact that only few symptomatic and no causative therapies are currently available. Despite considerable efforts in research, novel and effective approaches to treat PD have yet to be developed. Thus, new treatment strategies that slow the underlying disease are desperately needed. Potential neuroprotective and/or rescuing approaches have been proposed as an alternative to symptomatic treatment trying to suppress the possible causes of apoptosis of DArgic neurons. Recently, there has been a global trend towards the use of natural bioactive substances present in plants for the treatment of neurodegenerative disorders. Despite the great variety of plants in the world, only a few have had their pharmacological effects for antiparkinsonian activity and so, there are huge perspectives in this field for further research. Stem bark of Rhus verniciflua Stokes (RVS) has been used in herbal medicine and stomach disease for thousands of years in Korea in spite of containing urushiols, allergen in plant. Therefore, it is important to remove urushiol before using as a source of pharmaceutical compound. It has been reported that mushrooms play an important role in detoxification/removal of potential inhibitory phenolic compounds present in the substrate. Hence, we assumed that mushrooms may be used as a biological source for detoxification of RVS, not only because they are safe but also they contain various health enhancing substances. Therefore, in this study we have attempted to detoxify RVS by using mushrooms. In addition, despite the extensive research on several aspects of RVS, there is a paucity of knowledge about the therapeutic efficacy of RVS and detoxified RVS (DRE) (biologically removal of urushiols from stem bark of RVS using mushrooms) on neurodegenerative diseases. Thetrefore, this study was designed to evaluate the functions of RVS and of DRE in terms of the expression of catecholamine biosynthetic enzymes and neurotrophic factor encoding genes in rat and to demonstrate protective effects of RVS and DRE against oxidative insults-induced apoptosis in SH-SY5Y cells. First, we investigated the effect of DRE on viability of normal and tumor cells by 3- (4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results showed that DRE was not cytotoxic on human transformed primary embryonal kidney cell line, 293 and mouse fibroblast cell line, NIH3T3. The viability of both cells was increased significantly with low concentrations of DRE and conversely, it reduced cell proliferation and viability of most of the tested cancer /tumor cells in dose- and time -dependent manners. To evaluate the effect of DRE and RVS on catecholamine biosynthetic enzymes and neurotrophic factor genes expression, rats were gavaged with three concentrations of extract, 10, 20, and 40 ㎎/㎏ body weight, and euthanized 2, 4, 8, and 12 hr after dosing. Controls received no drug. Related mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR), and real-time PCR. Protein levels were measured by western blot and immunohistochemistry. Our results showed that both DRE and RVS induced tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC) and dopamine-β-hydroxylase (DBH) gene expression in rat brain. The induction of these enzymes was found to depend on the dose and duration of administration. Induction of TH, AADC, and DBH in response to DRE and RVS was biphasic, peaking at 2 and 4 hr. TH, AADC, and DBH also exhibited a transient induction by DRE and RVS; maximal at 2 hr. Longer term treatment for up to 12 hr leads to somewhat below control levels. In addition, our results showed that TH expression was up regulated at the low concentration of DRE and RVS for 2 hr and further increased was observed with the higher concentrations at 4 hr. We also found that the changes in TH and AADC proteins were co-related. Expression of TH and AADC is essential for phenotypic specification of DA neurons, while DBH expression is essential for the neurotransmitter phenotype of noradrenaline (NA) neurons. In this study, we detected consistent upregulation of TH and AADC mRNAs and proteins with RVS and DRE, but DBH was not expressed in brain by immunostaining and a small induction was observed by RT-PCR and western blot with DRE treatment and only by western blot with RVS treatment. These data suggest that TH-positive neurons induced by DRE and RVS are likely to be DA rather than NA. Although DBH was expressed in the locus coerulus, we could not detect the expression of DBH in DArgic neurons of the substantia nigra (SN). These results suggest that DRE and RVS have a complex and differential effects on TH, AADC, and DBH expression. Neurotrophic factors have attracted increasing attention because, in addition to promoting the survival and differentiation of developing neurons, they protect neurons against injury. In the present study, we have shown that oral administration of rats with DRE and RVS significantly increased the mRNA levels of brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF), and remarkably elevated the protein levels of these two neurotrophic factors in a dose- and time-dependent manner. Mitochondrial dysfunction and oxidative stress are suggested to be associated with the pathogenesis of PD. Accumulating evidences show that oxidative stress as one of the important pathways leading to neuronal cell death in PD. Excessive neuronal apoptosis may lead to massive neuronal tissue damage, as seen in some human neurodegenerative diseases, like PD. Therefore, the use of anti-apoptotic agents as a way of neuroprotection could be a potential therapy to slow or ameliorate the progression of neurodegenerative diseases. In the present study, we used SH-SY5Y cells to determine the protective effect DRE and RVS against rotenone, paraquat and 6-hydroxydopamine (6-OHDA)-induced toxicity. We found that DRE and RVS have significant neuroprotective effects against apoptosis induced by rotenone. Cellular survival was increased significantly in cells treated with these extracts prior to oxidative insults. We also demonstrated that DRE and RVS suppressed Fas, Caspase-3, Caspase-8, Caspase-9, and p53 activation. Therefore, these results suggest that DRE and RVS may act against rotenone neurotoxicity to inhibit apoptosis. In addition, a significant increase in TH and AADC protein was observed in SH-SY5Y cells with the treatment of rotenone alone. Interestingly, DRE and RVS treatment attenuated this novel compensatory contralateral increase in TH and AADC expression, presumably due to their neuroprotective effects. All the results in this study suggest that DRE and RVS may serve as an ideal adjuvant in regard of regulation of brain DArgic system and may contribute to neuroprotection in neurodegenerative diseases. Ultimately, elucidating the mechanisms by which DRE and RVS exerts their neuroprotective effects could provide novel therapeutic approaches in PD.