紅麴發酵產物改善帕金森氏症之研究

Abstract

現今社會人口逐漸趨向老化，相對地在醫療系統上老化疾病的治療需求也日漸攀升，較常見的老化疾病為阿茲海默症及帕金森氏症。帕金森氏症目前致病機轉雖尚未完全了解，但其患者多為腦中黑質緻密處之多巴胺神經元受損，使其無法產生多巴胺神經傳導物質，故此疾病治療方式為給予多巴胺前趨物 levodopa，但此藥物只在療程初期有效，因此發展新的有效成分應是保健食品改善帕金森氏症之重要研究方向。研究指出帕金森氏症患者腦中多巴胺神經元受損，其氧化壓力扮演重要角色，而抑制其氧化壓力所造成的細胞凋亡大多利用抗氧化物質來進行相關研究。紅麴米多年來為中國傳統醫藥及保健食品使用，由紅麴米可分離出高抗氧化能力的 dimerumic acid (DMA) 及 deferricoprogen (DFC)，以及可抑制細胞氧化壓力的 monascin (MS) 及 ankaflavin (AK)，故本研究評估紅麴發酵產物對帕金森氏症之改善效果。細胞試驗結果顯示，紅麴發酵產物中之 DMA 及 DFC 能使 6-hydroxydopamine (6-OHDA) 誘導 PC-12 細胞內 NADPH oxidase-2 (NOX-2) 蛋白表現量下降，使其胞內及胞外的活性氧自由基 (reactive oxygen species, ROS) 下降，且會透過調節 Bcl-2 associated X protein (Bax) 及 Bcl-2 蛋白質表現量，降低 caspase-3 的活性，使受損的分化後 PC-12 細胞凋亡數減少，達到保護神經細胞之作用。動物試驗以含有 DMA 及 DFC 之 50% 紅麴米酒精萃取物 (red mold rice extract with 50% ethanol, R50E) 來評估其對於 6-OHDA 誘導帕金森氏症大鼠之改善效果。實驗結果顯示，每 1 克 R50E 中含有 2.76 mg DMA 及 10.95 mg DFC。餵食 R50E (5.5 或 11.0 mg/kg) 可改善 6-OHDA 誘導帕金森氏症大鼠之運動障礙，並降低腦中黑質緻密處多巴胺神經元之衰退。R50E 可藉由提升腦中超氧歧化酶 (superoxide dismutase, SOD)、過氧化氫酶 (catalase, CAT)、穀胱甘肽還原酶 (glutathione reductase, GR) 及穀胱甘肽過氧化物酶 (glutathione peroxidase, GPx) 之活性，與調控 p47 phox、NOX1 及 NOX2 之 mRNA 表現量，來降低 6-OHDA 誘導帕金森氏症大鼠腦中活性氧自由基 (ROS) 及丙二醛 (malondialdehyde, MDA) 之含量。除此之外，R50E 亦可抑制 6-OHDA 誘導帕金森氏症大鼠腦中發炎因子一氧化氮 (nitric oxide, NO) 及腫瘤壞死因子 (tumor necrosis factors, TNF-α) 之含量。以上結果顯示，R50E 可透過抗氧化及抗發炎機制來防止大腦中多巴胺神經元之衰退，具有潛力應用於帕金森氏症之改善。DFC 及 DMA 亦可降低 6-OHDA 誘導大鼠中腦初代細胞 (mesencephalic neuron) 及 SH-SY5Y人類神經母細胞凋亡，DMA 可能透過調控 N-methyl-D-aspartate (NMDA) 受體及同型二聚體醣蛋白，DFC 則可能透過調控免疫球蛋白 Fc 受體，進而促進 protein kinase B (Akt) 及 extracellular signal-regulated kinases (ERK) 蛋白磷酸化，抑制 p38 及 c-Jun N-terminal Kinase (JNK) 蛋白活化，此外，DMA 及 DFC 亦可提升 heme oxygenase-1 (HO-1) 蛋白質表現，使受損 SH-SY5Y 神經細胞凋亡數減少，達到保護神經細胞之作用。

Society population is gradually moving towards aging. Relatively, demand for the treatment of aging diseases on the health care system has become more climb. The most common age-related neurodegenerative diseases are Alzheimer’s disease (AD) and Parkinson’s disease (PD). PD was found to be the loss of dopaminergic in the substantia nigra pars compacta (SNpc). Oral administration of levodopa remains the gold standard therapy for PD. But it is only effective for symptomatic relief during early stage of PD. There is therefore a great need to develop a new therapy for PD. Although the mechanisms responsible for dopaminergic death are not fully understood, accumulating evidence from studies suggests that oxidative stress plays the key role in initiating this cell death process. The presence of antioxidants protected against neuronal degeneration in dopamine neuron. Monascus purpureus-fermented rice, a traditional Chinese medicine as well as health food, includes multifunctional metabolites. M. purpureus-fermented products containing antioxidants: dimerumic acid (DMA) and deferricoprogen (DFC). We also found anti-oxidative stress compound-monascin (MS) and ankaflavin (AK) in Monascus- fermented secondary metabolites. Hence, the study used Monascus-fermented secondary metabolites to evaluate the improving effect on PD. In vitro, DMA and DFC reduced 6-hydroxydopamine (6-OHDA)-induced formation of extracellular and intercellular reactive oxygen species (ROS) and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2 associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Therefore, DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. In vivo, the present study was designed to investigate the effects of antioxidant-containing M. purpureus NTU 568-fermented rice extract (extracted with 50% ethanol, so called R50E) in a 6-OHDA-induced neurotoxicity in rats. R50E contained 2.76 mg of DMA and 10.95 mg of DFC per 1 g of freeze-dried extract. Administration of R50E (5.5 or 11.0 mg/kg) reduced parkinsonian motor dysfunction and the number of tyrosine hydroxylase (TH)-immunoreative neurons present in 6-OHDA-induced lesioned rats. Moreover, administration of R50E reversed the elevation of ROS and malondialdehyde (MDA) levels and promoted the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase via down-regulation of p47 phox, NOX1, and NOX2 expression in the 6-OHDA-lesion rats. Furthermore, treatment with R50E attenuated nitric oxide (NO) and tumor necrosis factors (TNF-α) levels in the 6-OHDA-lesion rats. In conclusion, R50E may prevent neurodegeration via anti-oxidative and anti-inflammatory mechanisms, suggesting its potential therapeutic value for PD treatment. DMA and DFC also inhibited 6-OHDA-induced apoptosis in mesencephalic neurons and SH-SY5Y cells. The protect effects of DMA and DFC may result from activation of protein kinase B (Akt) and extracellular signal-regulated kinases (ERK) pathway and inhibited the phoshorylation of p38 and c-Jun N-terminal Kinase (JNK) pathway. In addition, DMA and DFC may regulate N-methyl-D-aspartate (NMDA) receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor gene, respectively. These results suggested that the neuroprotection elicted by DMA and DFC against 6-OHDA-induced neurotoxicity was associated with Akt, MAPK, and heme oxygenase-1 (HO-1) pathway through NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc receptor.