J Nat Prod.2016 Jun;79(6):1604-1609

Daphnane Diterpenes from Daphne genkwa Activate Nurr1 and Have a Neuroprotective Effect in an Animal Model of Parkinson’s Disease. 

Baek-Soo Han,†, ‡ Kyoung-Shim Kim,‡, § Yu Jin Kim,^ Hoe-Yune Jung, Young-Mi Kang,§ Kyu-Suk Lee, Mi-Jin Sohn,^ Chun-Hyung Kim,êê, Ñ  Kwang-Soo Kim, êê and Won-Gon Kim*,^

 

Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea

  • Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea

^Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea

êêMolecular Neurobiology Laboratory, McLean Hospital and Program in Neuroscience, Harvard Medical School, Belmont, MA 02478

ÑPaean Biotechnology Inc., Daejeon 34031, Republic of Korea

These authors contributed equally to this work.

 

ABSTRACT:

Nurr1 is an orphan nuclear receptor that is essential for the differentiation and maintenance of dopaminergic neurons in the brain, and is a therapeutic target for Parkinson’s disease (PD). During the screening for Nurr1 activators from natural sources using cell-based assay systems, a methanol extract of the combined stems and roots of Daphne genkwa was found to activate the transcriptional function of Nurr1 at a concentration of 3 μg/mL. The active components were isolated and identified as genkwanine N (1) and yuanhuacin (2). Both compounds 1 and 2 significantly enhanced the function of Nurr1 at 0.3 mM. Nurr1-specific siRNA abolished the activity of 1 and 2, strongly suggesting that transcriptional activation by 1 and 2 occurred through the modulation of Nurr1 function. Additionally, treatment with 1 and 2 inhibited 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and lipopolysaccharide (LPS)-induced neuroinflammation. Moreover, in a 6-OHDA-lesioned rat model of PD, intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits and reduced tyrosine hydroxylase (TH)-positive dopaminergic neuron death induced by 6-OHDA injection, and had a beneficial effect on the inflammatory response in the brain. Accordingly, compounds 1 and 2, the first reported Nurr1 activators of natural origin, are potential lead compounds for the treatment of PD.

PMID: 27228307

 

Supplement :

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc), and is the most prevalent movement disorder, affecting 1–2% of the global population over the age of 65. Despite extensive research attempting to treat or delay the progress of PD, currently available pharmacological treatments largely address the symptoms, and almost all show severe side effects, such as dyskinesia.

Nurr1, an orphan nuclear receptor, is well-known as a key regulator of DA neuronal differentiation, and is also essential for the maintenance of DA neurons.1 Postmortem PD studies have shown diminished expression of Nurr1 in the brain. Recently, other roles of Nurr1 have received attention. In microglia and astrocytes, Nurr1 inhibits the expression of pro-inflammatory genes, together with repressor element-1 silencing transcription (REST) corepressor (CoREST), and the repression of inflammation by Nurr1 activation decreases PD symptoms and DA neuronal loss in both in vivo and in vitro PD models. Thus, activation of Nurr1 could improve the pathogenesis of PD. Indeed, very recently, small-molecule Nurr1 agonists such as amodiaquine have been reported to improve behavioral deficits in animal models of PD.2

Daphne genkwa Siebold et Zucc. (Thymelaeaceae) is widely distributed in Korea and mainland China, and is a well-known traditional oriental medicine. The flower buds of this plant (“Genkwa Flos”) are mainly used for diuretic, antitussive, expectorant, and antitumor purposes. Chemical investigation of D. genkwa has resulted in the isolation of flavonoids, coumarins, amides, and daphnane-type diterpenes. Daphnane-type diterpenes have been reported to show various biological activities, including antineoplastic, contraceptive, and neurotrophic activities.

During the course of screening for Nurr1 activators from Korean medicinal plants, the methanol extract of the stem and root portion of D. genkwa was found to have potent Nurr1-activating activity, as well as neuroprotective effects, in an animal model of PD.

 

 

Figure. Discovery of Nurr1 activator from traditional oriental medicine

 

Two active components, genkwanine N (1) and yuanhuacin (2), were isolated from the hexane extract using bioassay-guided fractionation, and were identified as genkwanine N and yuanhuacin, respectively. Both compounds activated Nurr1 at 0.3 mM. A cell-based luciferase reporter assay in the absence or presence of Nurr1 specific siRNA was performed to investigate whether the induction of reporter gene expression by 1 or 2 was abolished by Nurr1 knockdown. Nurr1 knockdown by the addition of siRNA reduced 1– and 2-induced luciferase reporter gene activation by up to 83%. The knockdown of Nurr1 expression by the siRNA treatment was confirmed by Western blotting analysis. This data indicated that Transcriptional activation by 1 and 2 was mediated through the modulation of Nurr1 function.

Consistent with the fact that Nurr1 plays an important role in inflammation in microglia and astrocytes, 1 and 2 prominently reduced the expression of proinflammatory genes including interleukin-1β (IL-1β) and interleukin-6 (IL-6). Importantly, in a 6-OHDA-lesioned rat model of PD administrated with extremely low dose of 2 (0.5 mg/kg/day), d-Amphetamine-induced ipsilateral rotations were significantly decreased on day 14 and 42 following 6-OHDA lesion induction in the 2-treated group when compared with the vehicle-treated group..

Although daphnane-type diterpenes are the important compounds for the biological activities of D. genkwa, their in vivo toxicity has not been studied yet. Recently, total diterpenoids extract, composed of seven daphnane-type diterpenes (yuanhuacine, yuanhuadine, yuanhuafine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1), from Genkwa Flos was reported to show no lethal effect at 0.1 g/kg, but toxicity above 0.25 g/kg after oral administration to rats. Interestingly, the pharmacokinetic study suggested that yuanhuafine and yuanhuapine among the seven diterpenes were the key toxic components of D. genkwa in vivo.3

 

In summary, the present study shows that a methanol extract of the combined stems and roots portion of D. genkwa potently activated the transcriptional function of Nurr1, as well as improved behavioral deficits in a 6-OHDA-lesioned rat model of PD. The active principles were identified as 1 and 2, which enhanced the function of Nurr1 at 0.3 mM and ameliorated 6-OHDA-induced neuronal death and lipopolysaccharide-induced neuroinflammation in vitro. Importantly, the intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits in the animal model of PD, and reduced both tyrosine hydroxylase-positive dopaminergic neuron death and microglial inflammation in the 6-OHDA-lesioned striatum. Compounds 1 and 2, the first Nurr1 activators of natural origin, can therefore be suggested as promising novel lead compound candidates for the chemotherapeutic treatment of PD.

 

Acknowledgement: This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (114145-3). Support was also obtained from the KRIBB Research Initiative Program, Republic of Korea.

 

References

  1. Zetterstrom, R. H.; Solomin, L.; Jansson, L.; Hoffer, B. J.; Olson, L.; Perlmann, T. Science 1997, 276, 248-250.
  2. Kim, C. H.; Han, B. S.; Moon, J.; Kim, D. J.; Shin, J.; Rajan, S.; Nguyen, Q. T.; Sohn, M.; Kim, W. G.; Han, M.; Jeong, I.; Kim, K. S.; Lee, E. H.; Tu, Y.; Naffin-Olivos, J. L.; Park, C. H.; Ringe, D.; Yoon, H. S.; Petsko, G. A.; Kim, K. S. Proc. Natl. Acad. Sci. USA 2015, 112, 8756-8761.
  3. Chen, Y. Y.; Guo, J. M.; Qian, Y. F.; Guo, S.; Ma, C. H.; Duan, J. A. Phytomedicine 2013, 21, 82-89.

 

Contact:

Won-Gon Kim, Ph.D Principal Research Scientist

Superbacteria Research Center Korea Research Institute of Bioscience and Biotechnology 52 Eoeundong, Yusong, Daejeon 305-806, Republic of Korea E-mail) wgkim@kribb.re.kr

 

 

Figure 1. Activation of Nurr1 with compounds 1 and 2. (A) Luciferase assay to measure Nurr1 activity with 1, (B) with 2. Experiments were performed with human neuroblastoma SK-N-BE(2)C cells transfected with pTH2600LUC, pCMV Nurr1-myc, pZeoSV-Nurr1(LBD), and p8xGAL-Luc for the luciferase assay. (C) Luciferase assay to measure Nurr1 activity with 1 and 2 at 0.3 mM in Nurr1-knockdown cells. Human neuroblastoma SK-N-BE(2)C cells were transfected with pTH2600LUC, pCMV Nurr1-myc, pZeoSV-Nurr1(LBD), p8xGAL-Luc, and Nurr1-siRNA for the luciferase assay. (D) Western blotting analysis of Nurr1 in Nurr1-siRNA- treated SK-N-BE(2)C cells.

 

 

Figure 2. Inhibition of 6-OHDA-induced cell death by treatment on SH-SY5Y cells with compound 1 or 2. (A) Cellular viability assay with 6-OHDA in the presence or absence of 1 and 2 in SH-SY5Y cells (ANOVA and Turkey-Kramer method, n = 4; * p < 0.05, ** p < 0.01). (B and C) Western blotting analysis of cleaved caspase-3 and the release of mitochondrial cytochrome c into the cytosolic fractions during 6-OHDA-induced cell death with or without 1 and 2 at 6 mM. Actin and SOD-1 showed relatively equal protein loading in each lane.

 

 

 

Figure 3. Anti-inflammatory effects of 1 and 2 in a microglia cell line. (A) Analysis of IL-1β and IL-6 mRNA expression in LPS-treated BV-2 cells with or without compounds 1 and 2 at 3 mM (ANOVA and Turkey-Kramer method, n = 4; * p < 0.05, ** p < 0.01). (B) Western blotting analysis of IL-1β in LPS-induced inflammatory cells with or without 1 and 2 at 3 mM.

 

 

 

Figure 4. Effects of 2 on the behavioral asymmetry and dopaminergic cell death in a 6-OHDA-lesioned rat model of PD. (A) Experimental design. 6-OHDA-lesioned rats were administered vehicle or 2 for 2 weeks. Rotational behavior was tested at 2 and 6 weeks following 6-OHDA lesion indution. (B) d-Amphetamine-induced ipsilateral rotations in the two groups (Student’s t-test, *p < 0.05 and **p < 0.01). (C) Photomicrograph showing TH-immunoreactive cells in the SNc. Scale bar, 1 mm. (D) Percentage of SNc TH-positive cells in the lesioned side compared with those in the unlesioned side of vehicle-treated and rats treated with 2 (n = 10-14, Student’s t-test, *p < 0.05).

 

 

 

Figure 5. Effects of 2 on inflammation in a 6-OHDA-lesioned rat model of PD. (A) Photomicrograph showing Iba-1-immunoreactive cells in the dorsal striatum. Scale bar, 200 μm. (B) Number of Iba-1-positive cells in the lesioned striatum of vehicle-treated and 2-treated rats (n = 10-14, Student’s t-test, *p < 0.05).

 

 

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