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Medicinal herbs Oerianthe javanica (Blume) DC., Casuarina equisetifolia L. and Sorghum bicolor (L.) Moench protect human cells from [MPP.sup.+] damage via inducing FBX07 expression.


Background: The F-box protein 7 (FBX07) mutations have been identified in families with early-onset parkinsonism and pyramidal tract signs, and designated as PARK15. In addition, FBX07 mutations were found in typical and young onset Parkinson's disease (PD). Evidence has also shown that FBX07 plays an important role in the development of dopaminergic neurons and increased stability and overexpression of FBX07 may be beneficial to PD.

Purpose: We screened extracts of medicinal herbs to enhance FBX07 expression for neuroprotection in [MPP.sup.+]-treated cells.

Methods: Promoter reporter assay in HEK-293 cells was used to examine the cis/trans elements controlling FBX07 expression and to screen extracts of medicinal herbs enhancing FBX07 expression. MTT assay was performed to assess cell viability of [MPP.sup.+]-treated HEK-293/SH-SY5Y cells. In addition, proteasome activity, mitochondrial membrane potential and FBX07/TRAF2/GATA2 protein expression were evaluated.

Results: We demonstrated that -202--57 region of the FBX07 promoter is likely to contain sequences that are bound by positive trans protein factors to activate FBX07 expression and GATA2 is the main trans protein factor enhancing FBX07 expression. Extracts of medicinal herbs Oenanthe javanica (Blume) DC. (Umbelliferae), Casuarina equisetifolia L (Casuarinaceae), and Sorghum bicolor (L) Moench (Cramineae) improved cell viability of both [MPP.sup.+]-treated HEK-293 and SH-SY5Y cells, rescued proteasome activity in [MPP.sup.+]-treated HEK-293 cells, and restored mitochondrial membrane potential in [MPP.sup.+]-treated SHSY5Y cells. These protection effects of herbal extracts are acting through enhancing FBX07 and decreasing TRAF2 expression, which is probably mediated by GATA2 induction.

Conclusion: Collectively, our study provides new targets, FBX07 and its regulator GATA2, for the development of potential treatments of PD.



Promoter characterization

Medicinal herbs




Parkinson's disease (PD) is one of the most common neurodegenerative disorders with selected loss of the nigro-striatal dopaminergic neurons and multifactorial etiologies. Although PD is attributable to environmental factors, it is now known to associate with genetic factors. In the recent years, genetic studies have identified genes causing rare dominant or recessive monogenic forms of PD which include SNCA, Parkin, PINK1, DJ-1. LRRK2, ATP13A2, PLA2C6, VPS35, EIF4G1, SYNJ1, DNAJC6, and DNAJC13 (Trinh and Farrer, 2013). The functional studies on protein products of the genes and the pathogenetic effects consequent to their mutations suggest that accumulation of aberrant or misfolded proteins, mitochondrial dysfunction, increased oxidative stress, impaired ubiquitin-proteasome function, failure of autophagy-lysosome and mitophagy, and deficits in synaptic exocytosis, endocytosis, and endosomal trafficking are involved in pathogenesis of PD (Trinh and Farrer, 2013). Although mutations in these genes have also been found in some of late-onset sporadic PD, the majority of primary causes and treatments for PD remain to be explored.

The F-box protein 7 (FBX07) mutations (homozygous R378G, compound heterozygous IVS7 + 1 G/T & T22M, and homozygous R498X) have been found in families with early-onset parkinsonism with pyramidal tract signs (Paisan-Ruiz et al., 2010; Di Fonzo et al., 2009; Shojaee et al., 2008). Two missense substitutions, 187T and D328R, in a single heterozygous state, were identified in two early onset PD patients in Taiwan (Lin et al.. 2013). Recently, a new homozygous FBX07 gene mutation, L34R causing typical PD was reported (Lohmann et al., 2015). FBX07 is a member of the F-box-containing protein (FBP) family and characterized by approximately 40 amino acid motif, the F-box (Ilyin et al., 2000). The F-box interacts with Skp1 protein. Therefore, FBP is a part of SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complexes and involved in ubiquitin-mediated proteasomal degradation (Ho et al., 2008). In addition, FBX07 binds to, and mediates ubiquitin conjugation to cIAP1 (cellular inhibitor of apoptosis protein 1) and TRAF2 (TNF receptor-associated factor 2), which leads to lowered activity of NF-[kappa]B (nuclear factor [kappa]B) (Kuiken et al., 2012). Additionally, FBPs might also function through non-SCF mechanisms, in processes such as transcription, cell cycle regulation, mitochondrial dynamics and intracellular trafficking (Nelson et al., 2013). More evidence has shown that FBX07 participates in integrity of mitochondrial function through direct interaction with PINK1 and parkin and plays a role in parkin-mediated mitophagy. Overexpression of Fbxo7 could rescue loss of parkin in a Drosophila model, suggesting a functional interaction between the two proteins (Burchell et al., 2013). Taken together, FBX07 plays an important role in ubiquitin-proteasomal pathway, NF-[kappa]B signaling, and mitochondrial function.

We have previously reported that variant Y52C exerts a protective effect on Taiwanese PD through increasing stability of FBX07 protein and decreasing TRAF2 level (Chen et al., 2014). Reduced expression of FBX07 has been shown to cause dysfunction of dopaminergic neuron and locomotor defects in zebrafish, suggesting that FBX07 contributes to the integration of dopaminergic neurons and its loss of function resulted from mutations may lead to the manifestation of PD (Zhao et al., 2012). As the protective role of increased FBX07 level is evident and overexpression of FBX07 may be beneficial to PD, we aim to characterize the eis and trans-acting elements in promoter controlling FBX07 expression and to screen medicinal herbs that may enhance FBX07 expression as potential treatments for PD.

Materials and methods

Herbal extract preparation and HPLC analysis

Fourteen collected medicinal herbs (Table 1) were cleaned with water, dried at 55 [degrees]C overnight, then chopped into pieces or powdered with grinder as the raw materials. The oven-dried raw materials were extracted in 95% ethanol at a ratio of 1:10 (w/v) on a rotary shaker at 150 rpm for one week. After extraction, the mixture was filtered with Whatman No. 1 filter paper, evaporated to a final volume around 30 ml, then lyophilized to obtain the tested herbal extracts, and stored frozen at -20 [degrees]C. From 100 g of dry herbal material, approximate 0.5-4.9 g of dry extract was obtained (Table 1). The chromatographic separation of the tested herbal extracts (10 [micro]l, 50mg/ml) was carried out on a Hitachi LaChrom Elite HPLC system, consisting of a HTA pump (L-2130), an auto-sampler (L-2200), a column oven (L-2350), and a photo diode array detector (L-2455). The column used was a Hypersil ODS (C18) column (250x4.6 mm I.D., 5 [micro]M, Thermo). The mobile phases are composed of distilled water contains 0.1% phosphoric acid (A) and acetonitrile (B), followed by gradient elution of 20-60% B (v/v) 0-40 min, 60-90% B (v/v) 40-45 min, 90% B (v/v) 45-55 min, 90-20% B (v/v) 55-60 min, 20% B (v/v) 60-70 min at 30 [degrees]C. The flow rate was 0.8ml/min. The UV was monitored at 368 nm. Given that quercetin, apigenin and kaempferol found in many plants are potent antioxidants that could play important roles in treating neurodegenerative diseases including PD and Alzheimer's disease (Filomeni et al., 2012; Han et al., 2012; Pandey et al., 2012; Zhao et al., 2013a), the three natural flavonols (5 [micro]l, 0.25 mM; Sigma) were used as reference compounds for further quality control (Supplementary Fig. S1).

FBX07 promoter and GATA1/2 cDNA plasmids

The FBX07 distal promoter fragment from -1240 to +261 (+1 represents the first nucleotide to be transcribed) was amplified using sense 5'-ATTAATTGCAGAATCAAGGGCAGGAT (Asel site underlined) and antisense 5'-GGCCTGGACCCGGAAGCG primers. The amplified FBX07 distal promoter fragment was cloned into [micro]gEMT Easy (Promega, Madison, WI, USA) and sequenced. Additional sense primers were designed to amplify FBX07 proximal promoter fragments from -694, -538, -438, -202, and -256 to +261. The cloned FBX07 promoter fragments were removed with AseI and EcoRI (in [micro]gEM-T Easy) and inserted upstream of the enhanced green fluorescent protein (GFP) gene in pEGFP-N1 (Clontech, Mountain View, CA, USA) to generate FBX07-GFP plasmids. The plasmids containing Flag-tagged human GATA binding protein 1 (GATA1) and GATA binding protein 2 (GATA2) in pCMV6-Entry vector were obtained from OriGene Technologies (Rockville, MD, USA).

Cell culture and transfection

Human embryonic kidney (HEK)-293 and 293T cell lines (ATCC No. CRL-1573 and CRL-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS). For FBX07-GFP transfection. 293T cells were seeded into 12-well plates with [10.sup.6] cells/well and transfected by T-Pro reagent (JF Biotechnology, New Taipei County, Taiwan) with 3 [micro]g FBX07 promoter plasmids and 1 [micro]g pDsRed-Monomer-C1 as a transfection control. For GATA1/2 cDNAs transfection, 293T cells were transfected with GATA1 and/or GATA2 cDNA plasmids (3 [micro]g total). Two days after transfection, the cells were harvested for further analysis. Human neuroblastoma SH-SY5Y cells (ATCC No. CRL-2266) were cultured in DMEM F12 supplemented with 10% FBS. All cells were maintained at 37 [degrees]C in a humidified incubator with 5% C[O.sub.2.]

FBX07 promoter activity assay

Two days after transfection with FBX07-GFP plasmids, cells were stained with Hoechst 33342 (0.1 [micro]g/ml, Sigma, St. Louis, MO, USA) and the fluorescence colors were analyzed simultaneously using high-content analysis (HCA) system (ImageXpressMICRO, Molecular Devices, Sunnyvale, CA, USA), with excitation/emission wavelengths at 482/536 (GFP), 543/593 (DsRed) and 377/447 nm (Hoechst 33342). For GATA1/2 cDNAs transfection, cells were collected and analyzed for endogenous FBX07 and GAPDH (as an internal control) as well as transiently expressed GATA1-Flag and GATA2-Flag proteins (detected with Flag antibody) as described in Western blot analysis.

Chromatin immunoprecipitation-PCR

Using a ChIP kit (Millipore), about [10.sup.7] HEK-293 cells were cross-linked with 1% formaldehyde and resuspended in lysis buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris pH 8.1. Chromatin was sonicated to reduce the size of DNA to 200-2000 bp and centrifuged to remove cell debris. Sheared chromatin DNA mixture was incubated with 4 [micro]g GATA2 antibody or 2 [micro]g rabbit IgG (as a negative control) and protein A magnetic beads at 4 [degrees]C for 4 h with rotation. After washing beads several times, the attached immune complexes were eluted. The DNA was purified and analyzed by PCR using the primer pairs specific to the FBX07 promoter -198--80 (5'-GGGAGTGGCGACTGTATTGT and 5'-CTGGCATCCCAGATTTTCC, 119-bp fragment) and -142-+27 (5'-CACGCACACAGGGAGTAAGT and 5'-CTAAAGGAACCGGGCCTCTC, 169-bp fragment) regions.

Flp-In 293 FBX07 reporter cells and fluorescent assay

The FBX07 -694-+261 proximal promoter was used to construct Flp-ln 293 fluorescent reporter cells. The fragment containing the FBX07 promoter-driven GFP was excised with Asel and NotI restriction enzymes and used to replace an Asel-NotI fragment in pcDNA5/FRT/TO plasmid (Invitrogen, Carlsbad, CA, USA). The resulting FBX07 fluorescent reporter plasmid was used to generate Flp-ln fluorescent reporter cells and maintained according to the supplier's instructions (Invitrogen). Cells were plated into 96-well (2 X [10.sup.4]/well) plates, grown for 48 h and treated with different concentrations of the tested herbal extracts (Table 1, l-10 [micro]g/ml) for 24 h Then cells were stained with Hoechst 33342 and fluorescence was assessed by HCA system as described.

Western blot analysis

Total proteins were extracted using lysis buffer containing 10 mM Tris-HCI pH7.5. 150 mM NaCI, 5mM EDTA pH8.0, 0.1% SDS. 1% sodium deoxycholate, 1% NP-40 with protease inhibitor mixture (Sigma). Proteins (20 [micro]g) were heated in a boiling water bath for 10 min then fractionated by 10% SDS-polyacrylamide gel electrophoresis (PAGE). The fractionated protein samples were transferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA), and non-specific binding was blocked in 5% non-fat dry milk for overnight at 4 [degrees]C. After blocking, the blots were probed with FBX07 (1:1000 dilution, Abnova, Taipei, Taiwan), Flag (1:1000 dilution, Sigma), TRAF2 (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GATA1 (1:1000 dilution. GeneTex, Irvine, CA, USA), GATA2 (1:1000 dilution, GeneTex), or GAPDH (1:1000 dilution, MDBio, Taipei, Taiwan) at 4 [degrees]C overnight. After extensive washing, the immune complexes were detected by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibody (1:5000 dilution, GeneTex) and chemiluminescence substrate (Millipore, Temecula, CA, USA).

[MPP.sup.+] treatment and cell viability assay

HEK-293 and SH-SY5Y cells were plated into 6-well ([10.sup.6]/well) or 48-well (5 x [10.sup.4]/well) plates, grown for 16 h and treated with NTNU-319, 379, 395 or 439 (10 [micro]g/ml) for 8h Freshly prepared mitochondrial inhibitor [MPP.sup.+] (l-methyl-4-phenylpyridinium ion) was then added (200 [micro]M for HEK-293 or 3mM for SH-SY5Y cells) or not for 24 h At the end, 100 [micro]l MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5mg/ml in PBS, Sigma] was added to cells and incubated for 2 h at 37 [degrees]C in a humidified chamber. The absorbance of the insoluble purple formazan product was measured at 570 nm by a Bio-Tek [micro]Quant Universal Microplate Spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA).

Proteasome activity assay

HEK-293 cells were plated and treated with the tested herbal extracts and with or without [MPP.sup.+] as described. For proteasome activity assay, cells were collected and lysed in 0.5% NP40 in PBS. Lysate was sonicated and centrifuged at 10,000 g for 10 min at 4 [degrees]C. After quantitation, 20 [micro]g protein of cell lysates were transferred to a 96-well microtiter plate and incubated with fluorogenic substrate for measuring chymotrypsin-like activity of the proteasome according to the supplier's instructions (Abeam, Cambridge, MA, USA). Fluorescence (350 nm excitation, 440 nm emission) was measured on a microplate fluorometer (Infinite M1000, Tecan, Mannedorf, Switzerland) every 5 min for 1 h at 37 [degrees]C.

JC-1 mitochondrial membrane potential assay

JC-1 (5,5',6,6'-tetrachloro-1,1', 3,3'- tetraethylbenzimidazolylcarbocyanine iodide) was used to examine the mitochondria membrane potential ([DELTA][psi]m). JC-1 forms aggregates, which emit red fluorescence in the mitochondria of healthy cells. However, it remains as monomers that emit green fluorescence during the loss of [DELTA][psi]m. Briefly, SH-SY5Y cells were plated into 12-well (5 X [10.sup.6]/well) plates, grown for 16 h, treated with herbal extracts (lO [micro]g/ml) or kynurenic acid (KYNA) (10 [micro]M) for 8h, followed by treatment with or without [MPP.sup.+] (3 mM) for 24 h Then 500 [micro]l JC1 (5 [micro]M, Invitrogen) was added to cells and incubated for 30 min. The cells were collected, resuspended in PBS and analyzed using a flow cytometer (FACScan, Becton Dickinson, San Diego, CA, USA) at 488 nm excitation and 525 (green)/590 (red) nm emission. A total of [10.sup.4] cells were analyzed in each sample.

Statistical analysis

For each set of values, data are represented as mean [+ or -] SD of three independent experiments. Differences between groups were evaluated by two-tailed Student's t-test or ANOVA (one-way and two-way) with post-hoc LSD test where appropriate. P value less than 0.05 was considered statistically significant.


FBX07 promoter functional assay

Previously, a comparison of the porcine and human FBX07 promoter sequences revealed a high degree of sequence homology in the region -614-+10 relative to the transcription start site (Larsen and Bendixen, 2012). To study the regulation of human FBX07 promoter, 1.3-kb (-1240-1-261) to 0.3-kb (-56-+261) FBX07 promoter constructs (Fig. 1A) were prepared and tested in HEK-293T cell transfection assay. As shown in Fig. IB, GFP fluorescence reporter assay revealed that the FBX07 -694-+261 and -202-+261 promoter fragments displayed significantly higher promoter activity in comparison with -56-+261 fragment (119% and 129% vs.100%, P = 0.049 and 0.002, respectively). In addition, FBX07 -538-+261 and -438-+-261 promoter fragments displayed significantly lower promoter activity in comparison with -202-+261 fragment (111% and 98% vs.129%, P = 0.026 and <0.001, respectively). These results indicated positive and negative trans protein factors binding to -202--57 and -438--202 region, respectively to regulate FBX07 expression.

Protein factor modulating FBX07 expression

In silico searches of the -202--57 region of the FBX07 gene revealed conserved GATA1/2 binding motifs (Fig. 2A) (http:// and expression in brain and kidney ( GATA1 and GATA2 cDNAs were prepared and tested in 293T cell transfection assay. As shown in Fig. 2B, endogenous FBX07 levels in cells transfected with GATA1 and GATA2 cDNAs were 149% (P= 0.014) and 230% (P= 0.011) respectively of that in cells transfected with vector Plasmid alone. Co-transfection of GATA1 and GATA2 cDNAs further increased endogenous FBX07 expression to 267% (P = 0.008). Also endogenous FBX07 expression was significantly different between GATA1 and GATA2 (149% vs. 230%, P = 0.016), between GATA1 and GATA1/2 (149% vs. 267%, P= 0.001), but not between GATA2 and GATA1/2 (230% vs. 267%, P = 0.096) transfection. In vivo ChIP-PCR assay in 293T cells was also performed to examine the association between GATA2 and FBX07 DNA. Primers specific for the region flanking GATA2 binding sites amplified specific 119-bp (from -198 to -80) and 169-bp (from -142 to +27) products against background in chromatin precipitated by GATA2 antibody (Fig. 2C). The results suggest that GATA2 is the main trans protein factor modulating FBX07 expression in HEK-293T cells.

Screen of medicinal herbs enhancing FBX07 expression

It has been shown that FBX07 variant with increasing stability may contribute to reduced susceptibility to PD in Chinese population (Chen et al., 2014). Thus agents enhancing FBX07 expression may provide protective effect in PD. To screen potential medicinal herbs for enhancing FBX07 expression, we established a fluorescent reporter 293 cell model with the GFP reporter downstream of the FBX07 -694-+261 promoter (Fig. 3A). For drug screening with high content analysis, cells were treated with the herbal extracts (l-10 [micro]g/ml, Table 1) for 24 h followed by GFP fluorescence measurement (Fig. 3B). As shown in Fig. 3C, among the 14 tested extracts, six (NTNU-313, 319, 379, 395, 439 and 488) displayed potential of enhancing FBX07 promoter activity (105-118% in l-10 [micro]g/ml, P= 0.046-0.006). The enhancement effects of NTNU-319, 379, 395 and 439 (lO [micro]g/ml) on FBX07 expression in HEK-293 (113-121%, P= 0.036-0.015) and SH-SY5Y (115-117%, P = 0.039-0.021) cells were further confirmed by the Western blot after two days treatment (Fig. 3D).

Potential of medicinal herbs in cell survival upon [MPP.sup.+] treatment

To evaluate the cytotoxicity of NTNU-319, 379, 395 and 439, MTT assays were firstly performed on HEK-293 and SH-SY5Y cells after treatment with these herbal extracts (10 [micro]g/ml, Fig. 4A) for 32 h As shown in Fig. 4B, 97-102% of untreated HEK-293 cells for NTNU-319, 379, 395 and 439 (P>0.05) and 92-100% of untreated SH-SY5Y cells for NTNU-319, 379, 395 and 439 (P>0.05) were observed. To further examine the protection effect of the four herbs in [MPP.sup.+] -induced cellular models of PD, the mitochondrial inhibitor [MPP.sup.+] was applied to HEK-293 (0.2 mM) and SH-SY5Y (3.0 mM) cells for 24 h after 8 h of pre-treatment with NTNU-319, 379, 395 or 439. Both HEK-293 and SH-SY5Y cells displayed significantly decreased cell viability (HEK-293: 77%, P= 0.010; SH-SY5Y: 44%, P = 0.001) upon [MPP.sup.+] treatment, as compared to that of the untreated cells (100%). Conversely, on [MPP.sup.+] treatment, cell viability of herbal extracts-treated cells increased significantly compared to the untreated cells (HEK-293: 85-94% vs. 77%, P = 0.048-0.006; SH-SY5Y: 56-67% vs. 44%, P= 0.035-0.007) (Fig. 4B).

Medicinal herbs enhancing GATA2 expression to modulate FBX07 and TRAF2 abundance

Through binding and mediating ubiquitin conjugation, FBX07 has been shown to negatively regulate TRAF2 abundance (Chen et al., 2014; Kuiken et al., 2012). To assess the effects of tested herbal extracts on FBX07 and TRAF2 abundance, HEK-293 cells were treated with NTNU-319, 379, 395 or 439 (10ng/ml) alone or pretreated with herbal extracts for 8 h followed by [MPP.sup.+] treatment (0.2 mM) for 24 h Proteins were collected and blotted with TRAF2 and FBX07 antibodies. In addition, GATA1/2 expression levels were examined in these herbs pre-treated and/or [MPP.sup.+] treated cells. As shown in Fig. 4C, medicinal herb treatment alone did not significantly affect FBX07 (132-138%), TRAF2 (98-100%), GATA2 (103118%) and GATA1 (101-106%) expression levels (P>0.05). While [MPP.sup.+] treatment alone did not significantly affect FBX07, TRAF2, GATA2 and GATA1 expression levels (77-114% vs. 100%, P>0.05), endogenous FBX07 level was significantly increased with NTNU319, 395 or 439 pre-treatment (136-156% vs. 77%, P = 0.038-0.030) in [MPP.sup.+] cells. Accompanying that, TRAF2 protein was significantly decreased (74-46% vs. 107%, P = 0.030-0.003). In NTNU-319, 395 or 439 pre-treated cells, GATA2 (but not GATA1) protein level was significantly increased (120-148% vs. 100%, P = 0.027-0.019).

Through binding to proteasome inhibitor 31 (PI31), FBX07 acts as a regulator of proteasome (Nelson et al., 2013). Thus the effects of herbal extracts on proteasome activity upon [MPP.sup.+] treatment were also examined. As shown in Fig. 4D, when cells were treated with herbal extracts alone, 112-126% of untreated cells for NTNU-319, 379, 395 and 439 (P>0.05) were observed. [MPP.sup.+] treatment significantly reduced proteasome activity (52% vs. 100%, P = 0.009). Conversely, proteasome activity increased significantly in cells with NTNU-319, 395 or 439 pre-treatment (81-93% vs. 52%, P = 0.0120.004).

Protective effect of medicinal herbs on mitochondrial membrane potential

[MPP.sup.+] has been reported to specifically reduce mitochondrial potential (Lambert and Bondy, 1989). To assess the herbs' effect on mitochondria membrane potential upon [MPP.sup.+] treatment, SH-SY5Y cells were pre-treated with NTNU-319, 379, 395 or 439 (10 [micro]g/ml) for 8 h followed by [MPP.sup.+] treatment (3.0 mM) for 24 h Kynurenic acid (KYNA), a tryptophan metabolite known to own mitochondrial protection effect and attenuate [MPP.sup.+]-induced neuronal cell death in SH-SY5Y cells (Lee et al., 2008) was included as a positive compound for comparison. JC-1 fluorescence dye was used to measure the mitochondria membrane potential ([DELTA][psi]m) (Fig. 5A). As shown in Fig. 5B, when cells were treated with herbal extracts alone, percentage of cells with low [DELTA][psi]m changed from 34% for non-treatment to 31-32% for NTNU-319, 379, 395 and 439 (P> 0.05). Following treatment of [MPP.sup.+], percentage of cells with low [DELTA][psi]m increased (44% vs. 34%, P = 0.011). Conversely, percentage of cells with low [DELTA][psi]m decreased significantly with pretreatment of KYNA (36% vs. 44%, P = 0.021). This is also true for NTNU-319, 395 or 439 pre-treatment (36-32% vs. 44%, P = 0.023-0.011) (Fig. 5B and C).

Protective effects of apigenin on cell survival, GATA2/FBX07/TRAF2 expression, proteasome activity, and mitochondrial membrane potential

Thus upon [MPP.sup.+] treatment, NTNU-319, 395 and 439 induce FBX07 expression to protect human cells from [MPP.sup.+] damage. Among the three marker compounds examined, apigenin is present in all three herbal extracts, with 0.009%, 0.027% and 0.038%, respectively in 1 g/ml extracts, corresponding to 3.3 nM, 10.1 nM and 13.9 nM, respectively in 10 [micro]g/ml extracts. Apigenin was reported to display neuroprotective effects in [MPP.sup.+]/MPTP damaged PC12 cells (Liu et al., 2015)/mice (Patil et al., 2014). Protective effect of apigenin on cell survival was observed, with 1 [micro]M treatment in HEK-293 cells (78% vs. 71%, P = 0.029) or 0.1-1 [micro]M treatment in SH-SY5Y cells (71-74% vs. 66%, P = 0.011-0.006) (Fig. 6A). With 1 [micro]M apigenin pre-treatment, increased endogenous FBX07 (96% vs. 70%, P= 0.043) and GATA2 (113% vs. 75%, P= 0.016) and decreased TRAF2 (102% vs. 121%, P= 0.044) levels were observed in MPP^ cells (Fig. 6B). Accordingly, proteasome activity increased significantly in cells with 1 [micro]M apigenin pre-treatment (198% vs. 51%, P = 0.029) (Fig. 6C). Percentage of cells with low [DELTA][psi]m also decreased significantly with pre-treatment of 1 [micro]M apigenin (41% vs. 46%, P= 0.023) (Fig. 6D).


The decreased FBX07 protein in cells carrying FBX07 mutants has been shown (Zhao et al., 2011). The study of FBX07 knock down demonstrates that neuronal degeneration was attributable to decreased FBX07 in PD (Zhao et al., 2012). FBX07 was found to be co-localized in most of [alpha]-synuclein-positive inclusions in the postmortem brains of PD, implicating a role played by FBX07 in the pathogenesis of PD (Zhao et al., 2013b). Previously, we also showed that FBX07 variant resulting in increased protein stability and reduced TRAF2 may contribute to the reduced PD susceptibility (Chen et al., 2014). Recently, a study has shown an important role played by FBX07 in parkin-mediated mitophagy and maintenance of mitochondrial function, and overexpression of FBX07 rescues parkin mutant flies, which may suggest a neuroprotection role of FBX07 in PD (Burchell et al., 2013). However, how the FBX07 expression is regulated is not known. In this study using a FBX07 promoter functional assay, we have demonstrated that the -202-57 region of the promoter is likely to contain sequences that are bound by positive trans protein factors to activate FBX07 expression. Region between -438 and -202 may be bound by negative trans protein factors to lead to the observed lower promoter activity of -1240, -694, -538 and -438 fragments compared with -202 fragment. As highly sequence homology reported in the region -614-+10 between porcine and human FBX07 promoters (Larsen and Bendixen, 2012), more representative -694 instead of -202 fragment was used to screen potential medicinal herbs for enhancing FBX07 expression. We then showed that GATA2 is the main positive trans protein factor modulating FBX07 expression in HEK-293T cells. GATA2 is abundantly expressed in substantia nigra vulnerable to PD, and modulates SNCA expression in dopaminergic cells (Scherzer et al., 2008). Loss of GATA2 leads to severe defects in neurogenesis, suggesting the critical role of GATA2 in neuronal development (Nardelli et al., 1999). Our FBX07 promoter functional assay also implicates that agents inducing FBX07 expression may provide neuronal protection for PD by activating GATA2 expression. This was supported by the results that extracts of medicinal herbs Oenanthe javanica (Blume) DC., Casuarina equisetifolia L., and Sorghum bicolor (L) Moench improved cell viability of both [MPP.sup.+]-treated HEK-293 and SH-SY5Y cells, rescued proteasome activity in [MPP.sup.+] -treated HEK-293 cells, and restored mitochondrial membrane potential in MPP"-treated SH-SY5Y cells. As the results of our study have shown, these protection effects of herbal extracts are acting through enhancing FBX07 and subsequently decreasing TRAF2 expression, which is probably mediated by GATA2 induction. It is notable that although Flemingia philippinensis Merr. & Rolfe improved [MPP.sup.+] -induced cell toxicity in both HEK-293 and SH-SY5Y cells, it did not significantly affect the expression levels of FBX07, TRAF2, and GATA2, which suggests that this herbal extract may exert its cell protection effect through mechanism other than activating GATA2 and FBX07 expression. Indeed, Flemingia philippinensis Merr. & Rolfe has been shown to contain nine isoflavonerelated compounds with immunosuppressive activities (Li et al., 2011). Oenanthe javanica (Blume) DC. has been reported to have high antioxidant properties via increasing SOD1, SOD2, CAT, and GPx (Lee et al., 2015; Tae et al., 2014). A natural 3'-O-methylated flavonoid, isorhamnetin, from Oenanthe javanica (Blume) DC. was shown to have anti-inflammation effect through blocking of NFK [kappa]B activation (Yang et al., 2014). Persicarin from Oenanthe javanica (Blume) DC. also protects primary cultured rat cortical cells from glutamate-induced neurotoxicity (Ma et al., 2010). Casuarina equisetifolia (Blume) DC. leaves extract ameliorates gentamicin-induced nephrotoxicity and oxidative damage by scavenging oxygen free radicals (El-Tantawy et al., 2013). Sorghum bicolor (L.) Moench leaf sheath has been shown to protect rats against cisplatin-induced hepatotoxicity and nephrotoxicity and cyclophosphamide-induced neurotoxicity through reducing oxidative stress (Ademiluyi et al., 2014a, 2014b; Oboh et al., 2010). We further showed that apigenin, a common constituent in herbal extracts NTNU-319, 395 and 439, exerted its neuroprotective effect on MPTP-induced cytotoxicity through enhancing GATA2 and FBX07 and decreasing TRAF2 expression. It is notable that the concentrations of epigenin in herbal extracts used to achieve neuroprotective effect are lower than pure compound epigenin, which may be explained by the synergistic neuroprotection of multiple constituents in herbal extracts. Our study for the first time shows the protection effects of these herbal extracts on MPTP-induced cellular models of PD via inducing FBX07 expression, which may provide a new avenue for the development of therapeutics in PD. In addition, we also show direct evidence of interaction of GATA2 and the promoter region of FBX07 by using chromatin immunoprecipitation-PCR.


For the first time, our study shows that extracts of Oenanthe javanica (Blume) DC., Casuarina equisetifolia L., and Sorghum bicolor (L) Moench induce GATA2 expression, which may exert its trans effects on promoter of FBX07 to enhance FBX07 transcription, leading to decreased TRAF2, increased proteasome activities, restored mitochondrial membrane potential, and finally increased cell survival. This study provides evidence of these medicinal herbs as the potential treatments for PD.


Article history:

Received 17 August 2015

Revised 21 July 2016

Accepted 18 August 2016

Conflict of interest

The authors declare that there are no conflicts of interest.


We thank the Molecular Imaging Core Facility of National Taiwan Normal University for the technical assistance. This work was supported by the grants 104-2325-B-003-001 from the Ministry of Science and Technology, CMRPG3D005 and CMRPG3E142 from Chang Gung Memorial Hospital, and 103T3040B05 from National Taiwan Normal University, Taipei, Taiwan.

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.08.004.


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Chiung-Mei Chen (a,1), I-Cheng Chen (a,1), Ying-Lin Chen (b,1), Te-Hsien Lin (b), Wan-Ling Chen (a), Chih-Ying Chao (a), Yih-Ru Wu (a), Yeah-Ting Lu (b), Cheng-Yu Lee (c), Hong-Chi Chien (c), Ting-Shou Chen (d), Guey-Jen Lee-Chen (b), *, Chi-Mei Lee (b), *

(a) Department of Neurology. Chang-Cung Memorial Hospital Chang-Gung University College of Medicine, Taipei 10507. Taiwan (b) Department of Life Science. National Taiwan Normal University. 88 Ting-Chou Road, Section 4. Taipei 11677, Taiwan

(c) Center of Excellence for Drug Development. Industrial Technology Research Institute. Hsinchu 31040, Taiwan

(d) Center of Excellence for Diagnostic Products. Biomedical Technology and Device Research Laboratories. Industrial Technology Research Institute. Hsinchu 31040. Taiwan

Abbreviations: FBX07. The F-box protein 7: PD. Parkinson's disease; FBP. F-box-containing protein; SCF, Skpl-Cullinl-F-box protein; cIAPl. cellular inhibitor of apoptosis protein 1; TRAF2. TNF receptor-associated factor 2; NF-[kappa]B, nuclear factor [kappa]B; CFP, green fluorescent protein; GATA1, CATA binding protein 1; GATA2. CATA binding protein 2; HEK. human embryonic kidney; DMEM. Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HCA, high-content analysis; [MPP.sup.+] 1-methyl-4-phenylpyridinium ion; MTT, 3-(4.5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; JC-1, 5,5', 6.6'-tetrachloro-1,1', 3.3'-tetraethylbenzimidazolylcarbocyanine iodide; [DELTA] P[psi]m, mitochondria membrane potential; KYNA. kynurenic acid; PI31. proteasome inhibitor 31.

* Corresponding authors. Fax: +886 2 29312904.

E-mail addresses: (G.-J. Lee-Chen). (C.-M. Lee).

(1) These authors equally contributed to this work.


Table 1
The studied medicinal herbs.

Herb no.    Scientific name

NTNU-293    Euonymus laxiflorus Champ, ex Benth.
NTNU-301    Adiantum philippense L
NTNU-309    Hyptis brevipes Poit.
NTNU-312    Bischofia javanica Blume
NTNU-313    Sorbus randaiensis (Hayata) Koidz.
NTNU-319    Oenanthe javanica (Blume) DC.
NTNU-328    Cocculus orbiculacus (L) DC.
NTNU-331    Euonymus alacus (Thunb.) Siebold
NTNU-379    Flemingia philippinensis Merr. 8i Rolfe
NTNU-385    Prunus campanulata Maxim.
NTNU-395    Casuarina equisetifolia L.
NTNU-439    Sorghum bicolor (L.) Moench
NTNU-450    Phaseolus vulgaris L
NTNU-488    Platycladus orientalis (L) Franco

Herb no.    Family           Parts         Extraction yield (.%)

NTNU-293    Celastraceae     Stem          4.9
NTNU-301    Adiantaceae      Whole plant   2.2
NTNU-309    Labiatae         Stem          33
NTNU-312    Euphorbiaceae    Stem          1.5
NTNU-313    Rosaceae         Stem          1.4
NTNU-319    Umbelliferae     Whole plant   1.2
NTNU-328    Menispermaceae   Vine          2.0
NTNU-331    Celastraceae     Stem          1.4
NTNU-379    Leguminosae      Stem & root   1.9
NTNU-385    Rosaceae         Stem          1.1
NTNU-395    Casuarinaceae    Stem          0.5
NTNU-439    Gramineae        Seed          1.5
NTNU-450    Leguminosae      Seed          2.3
NTNU-488    Cupressaceae     Stem          1.8

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Title Annotation:Original article
Author:Chen, Chiung-Mei; Chen, I-Cheng; Chen, Ying-Lin; Lin, Te-Hsien; Chen, Wan-Ling; Chao, Chih-Ying; Wu,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Nov 15, 2016
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