PART 1: Cistanche Extracts Ameliorates The Neurotoxicity Induced By Hydrogen Peroxide in New Mutant DJ‐1‐transfected Neuroblastoma Cellular Models

Contact: joanna.jia@wecistanche.com

1. INTRODUCTION

The main neurodegenerative diseases include Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, and others. Among these disorders, the incidence rate of PD(Parkinson's disease) is second in the world and achieves 1%–2% in people over 60 years old (Burke & O'Malley, 2013; Mullard, 2017; Shen & Ji, 2013). As the major neurological characteristic of PD, the dopaminergic neurons in the substantia nigra pars compacta are progressive degenerative (Glizer & MacDonald, 2016), which is accompanied by the appearance of α‐synuclein inclusions called Lewy bodies (Zhang, An, Zhang, & Pu, 2010). In the pathogenesis of PD, rest tremors, rigidity, bradykinesia, and postural abnormalities are being diagnosed as the main symptoms of PD in the clinic. Although the exact progressive degenerative mechanisms of dopaminergic neurons are not understandable, either environmental causes including exposure to insecticides, neurotoxic agents, and heavy metals, or genetic causes such as mutations of Parkin (Kitada et al., 1998), α‐synuclein (Polymeropoulos et al., 1997), and DJ‐1 (Biosa et al., 2017; Bonifati et al., 2003), are believed to leading to the occurrence of PD. To date, the number of missense mutations, frameshift mutations, and large fragment deletions caused by DJ‐1 gene mutations exceeds 10 (Andres‐Mateos et al., 2007; Bonifati et al., 2003; Hague et al., 2003). Within DJ‐1 protein, the substitution of Leucine166Proline (L166P) stands out as important. In addition, it has also been shown that oxidative stress occurs in PD (Dexter et al., 1989, 1994; Nikam, Nikam, Ahaley, & Sontakke, 2009) and is involved in its pathogenesis (Hague et al., 2003).

DJ‐1 gene containing eight exons distributed over 24 kb, locates on chromosome 1p36 in humans (Bonifati et al., 2003), and encodes a highly conserved protein containing 189 amino acids. The protein belonged to the ThiJ/PfpI family, exists in a homodimer form. Glial cells within the cortex and substantia nigra and striatum are the main found in areas of DJ‐1 protein (Olzmann et al., 2007). In these cells, DJ‐1 plays the following roles, such as oncogene (Nagakubo et al., 1997), transcriptional regulation (Kim et al., 2005; Niki, Takahashi‐ Niki, Taira, Iguchi‐Ariga, & Agria, 2003), antioxidative stress (Taira et al., 2004; Zhou & Freed, 2005), chaperone (Shendelman, Jonason, Marlinat, Leete, & Abeliovich, 2004; Zhou, Zhu, Wioson, Petsko, & Fink, 2006), and protease (Abou‐Sleiman, Healy, Quinn, Lees, & Wood, 2003).

Treatment for neurodegenerative diseases: cistanche extract

Cistanche is traditional Chinese medicine and has been mainly used to treat andrology disease down the ages. The major active ingredients of Cistanche are phenyl glycosides including 34 compounds, such as acteoside, echinacoside, etc. The pharmacological effects of Cistanche extracts include improving sexual function, antiaging, increasing learning and memory ability, neuroprotection, immunomodulation, antifatigue, antiischemic, and liver protection (Tu et al., 2011). Caffeic acid is one of the major metabolites of Cistanche (Yan, 2018).

The commonly used cellular models of PD include 1‐methyl‐4‐ phenyl pyridinium ion‐induced PC12 cells (Abou‐Sleiman et al., 2003) and hydrogen peroxide (H2O2)‐induced neuroblastoma (SH‐SY5Y) cells (Zhang et al., 2009). However, the typical pathological features of PD are not present in these models. To develop a more physiologically relevant cellular model of PD, in this study we established H2O2 induced L166P and C106S DJ‐1‐transfected SH‐SY5Y cells, and investigated the effects of Cistanche extracts and key bioactive compounds, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides on these two models, for the first time.

Cistanche extract anti-neurodegenerative diseases

2. MATERIAL S AND METHODS

2.1 | Plasmids, drugs, chemicals, and cells

FLAG‐L166P DJ‐1, FLAG‐C106S DJ‐1 plasmids, and anti‐DJ‐1 poly‐ clonal antibodies were kindly supplied by Dr. Hiroyoshi Ariga, Graduate School of Pharmaceutical Sciences, Hokkaido University. Dehydrated minimal essential medium (MEM) and F‐12 medium were purchased from Gibco. Lipofectin was from Invitrogen. FastDigest Xho I and FastDigest EcoR I were from Fermentas. The endotoxin-free plasmid preparation kit was from biotech. The SH‐SY5Y cell line was from the Cell Bank of the Chinese Academy of Medical Sciences. BCA protein assay reagent kit was from Pierce. PVDF membranes were from Millipore. Anti‐FLAG polyclonal antibody was from GeneTex. Cistanche extracts, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides, were supplied by the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University.

2.2 | Plasmids amplification, extraction, and purification

After culturing Escherichia coli JM109 cells in LB liquid medium until OD600 = 0.5, competent cells were prepared using the calcium chloride method. The competent E. coli were then transformed with plasmid DNA using the heat shock method, after which the transformants were cultured on LB plates containing ampicillin for 16–24 hr at 37°C. A successfully transformed monoclonal colony was then selected and cultured in LB liquid medium containing 50 μg/mL ampicillin for an additional 12 hr at 37°C. The plasmids were extracted and purified using an endotoxin‐free plasmid preparation kit according to the manufacturer's instructions.

2.3 | Plasmid identification

Extracted plasmids were subjected to enzymatic digestion, after which the plasmids and digested fragments were examined using 1% agarose gel electrophoresis. The resultant gel was stained in EB solution for 20–25 min at room temperature and photographed.

2.4 | Plasmid transfection into SH‐SY5Y cells

Purified plasmid DNA was transfected into SH‐SY5Y cells cultured in MEM/F‐12 medium containing 10% fetal bovine serum. Transfected cells were then selected in a medium containing 400 μg/mL G418, after which 200 μg/mL G418 was used to maintain the stably transfected cell line.

2.5 | Identification of transfected cells by Western blot

Transfected SH‐SY5Y cells were lysed in RIPA buffer. After centrifugation of the lysate, the total protein concentration in the supernatant was determined using a BCA protein assay reagent kit. Equal amounts of protein extract were then subjected to a 12.5% SDS‐polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% nonfat dry milk in TBST solution and processed for immunodetection. Anti‐FLAG and anti‐DJ‐1 were used as primary antibodies, and HRP‐conjugated IgG was the secondary antibody. An enhanced chemiluminescence detection system was applied to detect the target proteins.

2.6 | Immunocytochemical identification of transfected cells

Transfected SH‐SY5Y cells were fixed with 4% paraformaldehyde solution, permeabilized with 0.3% Triton X‐100 solution, and blocked with 10% goat serum. The blocked cells were then incubated first with anti‐FLAG and anti‐DJ‐1 polyclonal antibody, and then with FITC‐conjugated goat antirabbit secondary antibody. The cell nuclei were stained with Hoechst 33342, and the cells were visualized under an inverted fluorescence microscope (IX‐71, Olympus).

2.7| Cell viability assay

Untransfected and transfected SH‐SY5Y cells were plated into 96‐ extracts, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides (all 10, 20, and 40 μg/mL, respectively), were added and the cells were incubated for 6 hr before treatment with 0.2 mM H2O2 for an additional 1 hr. The medium was discarded, and cell viability was assayed using MTT as described above.

2.8 | Effects of Cistanche extracts on cell viability

extracts, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides (all 10, 20, and 40 μg/mL, respectively), were added and the cells were incubated for 6 hr before treatment with 0.2 mM H2O2 for an additional 1 hr. The medium was discarded, and cell viability was assayed using MTT as described above.

2.9 | Statistical analysis

Data are expressed as the mean ± SD of three independent experiments. Differences between groups were analyzed using one‐way ANOVA and the LSD method with SPSS 22.0 software. Values of p < 0.05 were considered significant.

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3 | RESULTS

3.1 | Plasmids were successfully transformed and purified. After separate expression in E. coli JM109, plasmids were extracted and examined using 1% agarose gel electrophoresis after digestion with a restriction enzyme (Figure 1). The 6.2‐kb plasmids were cleaved into 5.4‐ and 0.8‐kb linear DNA fragments, which confirmed that the L166P and C106S DJ‐1 plasmids were both successfully expressed and purified.

3.2 | Identification of transfected cells by Western blotting. As shown in Figure 2, we detected FLAG‐L166P DJ‐1 and FLAG‐C106S DJ‐1 bands at about 70 kDa. The high levels of FLAG‐tagged protein in the transfected SH‐SY5Y cells indicate that the L166P and C106S DJ‐1 mutants were strongly expressed in their respective transfectants.

3.3 | Immunocytochemical identification of transfected cells. It shows the strong fluorescent signals from transfected SH‐ SY5Y cells after incubation with anti‐DJ‐1 or anti‐FLAG antibodies. This confirms the high levels of L166P and C106S DJ‐1 expressed in the transfectants.

3.4 | Transfected cells were more sensitive to H2O2 than untransfected cells. After incubation for 1 hr in the presence of graded concentrations of H2O2 (0.1, 0.2, 0.3, 0.4, 0.5, and 1 mM, respectively), the viabilities of SH‐SY5Y cells transfected with L166P or C106S DJ‐1 were dose-dependently reduced as compared to untransfected cells

3.5 | Cistanche extracts inhibited H2O2‐induced reductions SH‐SY5Y cell viability dose‐dependently inhibited by treatment with Cistanche extracts, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides (all 10, 20, and 40 μg/mL, respectively).

Cistanche