Echinacoside's nigrostriatal dopaminergic protection against 6-OHDA-Induced endoplasmic reticulum stress through reducing the accumulation of Seipin

Materials

ECH (CAS No. 82854-37-3, PubChem CID:5281771), 6-OHDA, DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), apomorphine, 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium (MTT) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 and foetal bovine serum were obtained from Gibco (Grand Island, NY, USA). The primary antibodies used in the study were as follows. Anti-seipin (Berardinelli–Seip congenital lipodystrophy 2, BSCL2) rabbit antibody was purchased from Abcam (Cambridge, MA, USA. Catalog: ab106793). Anti-seipin goat antibody was purchased from Santa Cruz (Dallas, TX, USA. Catalog: sc-55987). Anti-tyrosine hydroxylase (TH) antibody (ab75875), anti-GRP94 (ab108606) antibody, anti-GRP78/Bip antibody (ab108613) and anti-ATF4 antibody (ab50546) were purchased from Abcam. Anti-dopamine transporter (DAT) antibody was purchased from Proteintech (Wuhan, Hubei, China. Catalog number: 22524-1-AP). Anti-α-synuclein antibody (D37A6), anti-CHOP (L63F7) antibody and anti-ubiquitin (P4D1) antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies used in the study were as follows: donkey anti-goat IgG H&L (Alexa Fluor^® 488) (ab150129), goat anti-rabbit IgG H&L (Alexa Fluor^® 594) pre-adsorbed (ab150084), goat anti-mouse IgG H&L (HRP) (ab6789) and goat anti-rabbit IgG H&L (HRP) (ab6721), which were all purchased from Abcam. PureProteome™ Protein A/G Mix Magnetic Beads was purchased from Millipore (Darmstadt, Germany).

The BCA protein assay kit was purchased from Thermo Fisher Scientific (NJ, USA). Acetonitrile, methanol and phosphonate acid were all HPLC grade reagents purchased from Thermo Fisher, JC-1 and Mitochondrial Potential Sensors Kit (Invitrogen, Carlsbad, CA, USA).

Cell culture and treatment

PC12 cells were cultured in RPMI-1640 supplemented with 10% horse serum, 5% foetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C under a humidified atmosphere containing 5% CO₂. The cells were seeded into 96-well plates at a density of 1 × 10⁴/well. To determine the non-toxic dosages of ECH, PC12 cells were incubated with ECH (12.5–500 μM) for 24 hrs. To determine the toxic dosages of 6-OHDA, PC12 cells were treated with various concentrations of 6-OHDA (0–200 μM) for 24 hrs. To study the effect of ECH on cell viability in 6-OHDA-treated PC12 cells, after 80% confluence, the cells were pre-incubated with different concentrations of ECH (0, 25, 50, 100 μM) in a serum-free RPMI-1640 medium for 1 hr. Subsequently, 6-OHDA was added to the wells at a final concentration of 100 μM and incubated for another 24 hrs at 37°C.

Transfection with siRNA

To study the effect of seipin inhibition, PC12 cells were transfected with seipin siRNA (Cat. No: 133000; Thermo Fisher Scientific). Briefly, a monolayer of 1 × 10⁶ cells in a 6-well plate was transfected with 20 nM of siRNA using Lipofectamine2000™ (Life Technologies, Carlsbad, CA, USA). The cells were washed twice with phosphate-buffered saline (PBS) to remove serum and supplemented with serum-free medium containing a transfection cocktail. The transfected cells were incubated for 12 hrs followed by replacement of the growth medium containing the transfection cocktail with complete DMEM medium containing serum for an additional 10 hrs. The cells were then trypsinized, recounted and seeded at 2 × 10⁵ cells per well in 12-well plates. The following day, cells were treated with or without 6-OHDA to assess the silencing effect of specific targets on 6-OHDA-mediated ERS. The efficiency of gene silencing was assessed using Western blotting and found to be between 40% and 70%.

Cell viability assay

Cell viability was determined using an MTT assay 22. A total of 0.5 mg/ml MTT was added to each well after the initial incubation, and incubated for further 4 hrs at 37°C. After the supernatant was removed, the formazan product was solubilized with 100 μl dimethyl sulfoxide (DMSO), and optical density at 570 nm was measured using an Epoch microplate assay reader (Bio-Tek, Winooski, VT, USA).

Transmission electron microscopy observation of PC12 cells

Following each group treatment described in Section 2.2, PC12 cells were washed with ice-cold Ca²⁺-free PBS, re-suspended in PBS and centrifuged at 1000 r/min for 5 min. The cell precipitate was then fixed in 2.5% glutaraldehyde. After 90 min. of fixation, the cells were washed and post-fixed in 1% OsO₄ in a cacodylate buffer. The samples were then dehydrated with successively increasing concentrations of ethanol and embedded in Epon812. Thin sections were stained with uranyl acetate followed by lead citrate, and observed using a Hitachi HT-7500 (Tokyo, Japan) transmission electron microscope.

Measurement of intracellular ROS production using flow cytometry

Each group treatment described in ‘cell treatment’ section above washed with ice-cold Ca²⁺ free PBS, and re-suspended in PBS, centrifuged at 1000 r/min. for 5 min. The cells were centrifuged and washed twice with PBS. Cells were loaded with 10 μM H₂DCFH-DA for 30 min. at 37°C in the dark and analysed in a flow cytometer at 488 nm excitation.

Animal surgical operation

All animal studies were performed with the approval of the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine, and were performed according to the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals (NIH publication no. 80‑23). Male Sprague Dawley rats, weighing 200–220 g, were used in this study. Rat was housed individually in cages with food and water consumed ad libitum. Environmental conditions were strictly controlled, with a 12-hr light/dark cycle, maintained at 24°C and 50% humidity.

Fifty animals were randomly divided into five groups, consisting of control (n = 10), vehicle (n = 10), 6-OHDA (n = 10), and 6-OHDA plus ECH-high dosage (n = 10) and -low dosage (n = 10) groups. The control group did not undergo surgery. After anaesthesia with chloral hydrate (350 mg/kg), the skull was exposed and burr holes were drilled to introduce a syringe for injection (Fig. S1). The 6-OHDA group and 6-OHDA plus ECH group animals received 6-OHDA (8 μg/4 μl in 0.9% saline containing 0.1% ascorbic acid) injections into the right substantia nigra pars compacta (SNpc; A/P:−5.0 mm, L:+ 1.9 mm, V: −7.8 mm) and ventral tegmental area (VTA; A/P: −4.6 mm, L: +0.9 mm, V: −7.5 mm) 23 with a 5-μl Hamilton Syringe on stereotaxic apparatus (68002, RWD-Life Science, Shenzhen, China). All injections were administered at a rate of 0.5 μl/min. The needle was left in situ for an additional 5 min. before retraction of the needle at a rate of 1.0 mm/min. Post-surgery, with the skin sutured, the animals were removed from the stereotaxic instrument and kept under a heat lamp and a thermal blanket for 30 min. to maintain body temperature before being returned to their cage. Rats that showed positive results in a rotation test 3 weeks after surgery were selected as successful PD models. The vehicle group underwent the same procedures as the 6-OHDA group except for receiving injections of saline instead of 6-OHDA solution. Control, vehicle and 6-OHDA groups were administered 0.9% saline (2 ml/kg) intraperitoneally for the following 14 days. The 6-OHDA plus ECH-high and ECH-low dosage groups received ECH intraperitoneally at a dose of 7 and 3.5 mg/kg, respectively, for 14 days. The administration was carried out once a day at 8 o'clock a.m.

One day after the last injection, the rats were killed and the striatum and substantia nigra from five rats in each group were dissected and frozen in liquid nitrogen and stored at −80°C for analysis with high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS), Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Five animals per group were anaesthetized with chloral hydrate (350 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde for pathological and immunohistochemical (IHC) studies. The whole procedure of the animal experiments is provided in the supplementary materials (Fig. S2).

Rotation behavioural analysis

Rotation behavioural analysis was performed 3 and 5 weeks following surgery. Rats were injected with apomorphine (0.5 mg/kg, i.p.) and placed in a 40-cm-diameter stainless steel cylindrical bowl. Rotations over a 30-min. period beginning 10 min. after the administration of apomorphine were counted. Data are expressed as contralateral net turns/min and results with the number of total turns over 210 were recorded and analysed. Assessments were carried out by an observer who was blind to the animal pre-treatments.

Determination of dopamine and metabolite concentrations

The striatum of the rats was rapidly dissected and stored at −80°C before the quantification of DA, DOPAC and HVA using UHPLC-MS/MS. The experiments were performed using an Agilent 1290 ultra-high-performance liquid chromatography system consisting of a binary pump, autosampler and thermostated column compartment. The UPLC analyses were carried out using an elution profile composed of a first isocratic step of water (formic acid 0.1%), followed by acetonitrile (ACN) 95:5 for 0.5 min., and then 30% ACN for over 1 min. to separate DA, DOPAC and HVA at 25°C. The column was then washed with 90% ACN for 0.5 min., followed by equilibration of the column for 0.5 min. with 5% ACN. The flow rate was 0.3 ml/min. The injection volume was 5 μl. An Agilent 6460 triple quadrupole-mass spectrometer with an electrospray ion source operated in the positive mode was used for detecting DA and DOPAC. The negative mode was used for detecting HVA. Flow injection analysis was used to optimize the fragmentor and source parameters. The optimized source parameters for MS analysis were as follows: drying gas temperature, 350°C; gas flow, 10 l/min.; nebulizer gas flow pressure, 35 PSI; and capillary voltage, 4500 V. The optimized fragmentor voltage, collision energy and MRM pairs are reported in Table 1.

DA, DOPAC and HVA levels were calculated by extrapolating the peak area from a standard curve (ranging from 1 to 100 nM of mixed DA, DOPAC and HVA). The identification of peaks was carried out by comparison with standards. The amounts of DA, DOPAC and HVA in each sample were quantified by comparing the peak area of the samples to those of the standards. The units are expressed as ng/g of wet weight.

Pathologic and immunohistochemical detection

After the rats were deeply anaesthetized and transcardially perfused, the brain was removed from the skull and dehydrated while immersed in the fixative solution for 1 week at 4°C. Slices of interest were selected from the SN-VTA located −5.4 mm from bregma according to the atlas of Paxinos and Watson 23. Haematoxylin and eosin (HE) staining of paraffin-embedded sections from each rat, with a slice thickness of 4 μm, was used to locate the SN-VTA slices and observe neuronal morphology. The images were taken using an Olympus camera (DP72; Olympus, Tokyo, Japan) at an original magnification of 400×. Tissue sections were incubated with anti-TH antibody (1: 200) in a blocking solution (Triton X-100 0.4% v/v and NGS 3% v/v in PBS) for 1 h. After incubation, the slices were washed four times with PBS 0.01 M for 10 min each time. A second incubation with anti-rabbit antibody (1:2000) was performed for 2 hrs at 25°C. After several washings with PBS, the antibody complex was detected using a modification of the ABC system (Kit Vectastain ABC, Vector Laboratory Inc., Burlingame, CA, USA). The midbrain dopaminergic cell groups were plotted from TH-immunostained slices. TH-immunoreactive (TH-ir) neurons were stereologically quantified using Image-Pro Express 6 (Media Cybernetics, Rockville, MD, USA). The mean number of TH-ir neurons in each hemisphere, obtained through the quantification of four alternating slices, was considered to be representative of the SNpc neuronal cells in each animal. The selected areas were digitized with a microscope (DX45; Olympus). The images were taken using an Olympus camera (DP72; Olympus) at an original magnification of 100×.

For double immunofluorescence, the tissue sections were incubated with anti-TH antibody and diluted (1:200) and anti-BSCL2/Seipin antibody (1:200; Santa Cruz); TH was imaged with anti-rabbit IgG H&L (Alexa Fluor^® 594) (1:200; Abcam) and seipin by anti-goat IgG H&L (Alexa Fluor^® 488) (1:200; Abcam). The slides were mounted in a fluorescent mounting medium containing DAPI (Invitrogen). The sections were examined with a Zeiss Axiophot microscope equipped for epifluorescence illumination.

Western blotting and ubiquitination detection

PC12 cells and striatum lysates were prepared in an ice-cold lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, 0.1% SDS, 1 mM Na₃VO₄, 10 mM NaF and 1 mM EDTA) containing a 1% protease inhibitor cocktail (Sigma Chemical, St. Louis, MO, USA) and centrifuged at 16,000 g for 30 min. at 4°C. Next, 20 μg of total protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (10%) and proteins were transferred to a PVDF membrane, which was subsequently incubated in 0.01 M PBS, 0.1% Tween-20 and 5% non-fat dry milk powder to block any remaining protein binding sites. Membranes were incubated overnight at 4°C using the following primary antibodies: the rabbit antibody against TH (1:2000), rabbit antibody against DAT (1:1500), mouse antibody against GAPDH (1:1000), rabbit antibody against seipin (BSCL2), GRP94, GRP78/Bip, ATF4, CHOP, alpha-synuclein and ubiquitin antibody (1:1000). After three 5-min. washes in TBST, membranes were incubated with the goat anti-mouse or rabbit IgG (1:2000; Abcam) for 60 min. at room temperature. All antibody incubations and washing steps were carried out in 0.01 M PBS and 0.1% Tween-20. The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ChemiDoc XRS+, Bio-Rad, Hercules, CA, USA) and quantified with densitometry using ImageJ 1.49s software.

Detection of seipin ubiquitination followed by Yeun Su Choo's protocol 24 with little modification. Briefly, 1 × 10⁹ PC12 cells were harvested after treated with different conditions (6-OHDA, ECH and 6-OHDA plus ECH) were harvested and 1 ml complete cell lysis buffer of each group was collected to measure the protein concentration, to prepare Protein A/G Mix magnetic beads (Millipore) and anti-seipin antibody (5 μg) conjunction according to the instructions and to incubate the prepared beads and 1 mg protein lysis at 2–8°C overnight to capture protein compounds cross-linked with seipin. After immunoprecipitation and native elution, the samples were boil with 2 × SDS loading buffer, then load samples onto a SDS-PAGE gel for immunoblotting analysis. Seipin ubiquitination was detected by anti-ubiquitin antibody. Stripping membrane and staining Ponceau S was taken as loading control.

Quantitative real-time polymerase chain reaction

Frozen tissue samples were homogenized in 1 ml Trizol reagent, and total RNA was extracted following the manufacturer's protocol. After ethanol precipitation, the vacuum dried RNA was dissolved in 50 μl nuclease-free water and the concentration and purity of isolated RNA was determined using Nanodrop 2000 (Thermo Fisher). The 260/280 nM ratios of the samples were between 1.7 and 2.1. The integrity of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Degradation was defined as a shift in the RNA size distribution towards smaller fragments and a decrease in fluorescence signal of ribosomal peaks. Only samples with defined ribosomal peaks were used in this study. For cDNA synthesis, 500 ng total RNA was mixed with 5 μl RT master mix (PrimeScript™ RT Master Mix, Otsu, Shiga, Japan). RNase-free H₂O was added to reach a final total volume of 10 μl. The solution was incubated for 15 min. at 37°C and terminated by incubating for 5 sec. at 85°C. Distilled water was added to the reaction mixture to reach a final volume of 40 μl. Real-time qPCR was performed with an ABI 7500 Instrument (Bio-Rad Laboratories) using the DNA binding dye SYBR Green I for the detection of PCR products. PCRs were set up in 96-well plates using 2 μl cDNA with 10 μl 2× selected SYBR Green I master mix (life) containing forward and reverse PCR primers with RNase-free H₂O added to reach a final volume of 20 μl. The primer pairs used are listed in Table 2. Amplification was performed with a hot start polymerase activation step for 10 min. at 95°C, followed by 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. The cycle threshold (CT) values of the target and reference genes were identified. All samples were run three times. The CT values were normalized by subtracting the CT value of the housekeeping gene GAPDH from the CT value of the target gene (ΔCT). The fold change between groups was calculated using the CT value calculated using the method of 2^−∆∆Ct (∆CT = CT [target gene] – CT [GAPDH]). Samples with an average CT value less than 32 were considered eligible for analysis. All samples were run three times.

Statistical analysis

SPSS Version 19.0 for Windows (IBM Corp, Armonk, NY, USA) was used for all statistical analyses. All experiments were performed at least three times. Comparisons between two groups were performed using a two-samples Student's t-test, and comparisons between multiple groups were performed using a one-way ANOVA followed by Tukey's post hoc test. Data are expressed as mean ± S.D. P < 0.05 was considered statistically significant.

Protective effect of ECH on viability of PC12 cells injured by 6-OHDA

In this study, we identified that ECH (chemical structure shown in Fig. 1A, CAS No. 82854-37-3) has a protective effect on nigrostriatal dopaminergic neurons. To determine non-toxic dosages, we detected the cell viability of PC12 cells treated with different concentrations of ECH for 24 hrs, as shown in Figure 1B. Following treatment with concentrations of 12.5–200 μM of ECH for 24 hrs, no significant difference was seen in the viability of PC12 cells. However, with a concentration of 500 μM, cell viability was significantly lower than at other concentrations. Consequently, we chose to use concentrations of 25, 50 and 100 μM in the study.

We then examined PC12 cell viability following 6-OHDA treatment. As shown in Figure 1C, 6-OHDA reduced cell viability in a dose-dependent manner and cytotoxicity was significantly induced at concentrations ≥100 μM 6-OHDA (P < 0.01). Therefore, we used 100 μM 6-OHDA for 24 hrs as the optimal standard concentration and time course for the induction of cytotoxicity in the subsequent experiments.

To determine whether ECH exerted neuro-protective effects against the action of 6-OHDA, PC12 cells were pre-incubated with the concentrations of 0, 25, 50 or 100 μM ECH for 1 hr followed by exposure to 100 μM 6-OHDA for 24 hrs. The MTT assay showed that treating PC12 cells with 6-OHDA alone resulted in a 20% reduction in the number of surviving cells after 24 hrs. Co-treatment with 50 or 100 μM ECH showed a reduction in 6-OHDA-induced cytotoxicity (P < 0.05 versus 6-OHDA alone). PC12 cell viability increased in an ECH-dose-dependent manner compared with the cells treated with 6-OHDA alone. This suggests that ECH reduced 6-OHDA-induced cytotoxicity.

ECH protects against 6-OHDA-induced SNpc lesions in vivo

ECH maintained complete ultrastructure of endoplasmic reticulum damaged by 6-OHDA in vitro

To evaluate ER morphology transformation following 6-OHDA and ECH treatment, the ER ultrastructure was determined using transmission electron microscopy. The results show that 100 μM 6-OHDA-induced ER curling and generated a vacuole, shown in Figure 2A. The ECH-treated group showed recovery of ER structural integrity and caused extension of the ER. The ER is marked by a black arrow.

ECH protects against 6-OHDA-induced SNpc lesions in vivo

ECH reduced pathologic lesions and up-regulated tyrosine hydroxylase (TH) expression in SNpc following 6-OHDA treatment

To verify the neurotoxic effects of 6-OHDA and the protective effects of ECH, we observed pathological changes in the striatum using HE staining. The main morphological changes in the 6-OHDA-treated rat model in this study were characterized by white matter oedema in the striatum, enlargement of nerve fibres, and neuronal degeneration and necrosis in the SN. As showed in Figure 3A, abnormalities such as nerve cell degeneration, necrosis and white matter oedema, congestion in interstitial vasculature, infiltration of inflammatory cell and proliferation of glial cells were not observed in the control or vehicle groups. In the 6-OHDA and 6-OHDA plus ECH groups, white matter oedema in the striatum was seen. In the SNpc, part of the nerve cell structure was not clear, the nucleus was no longer apparent, and the cytoplasm had a granular appearance. The nuclei of individual cells were deeply stained and the size of the nuclei was reduced. However, in the 6-OHDA plus high-dose ECH group, there was a significant reduction in the extent of these changes compared with the 6-OHDA group.

We examined the extent of 6-OHDA-induced dopaminergic neuronal degeneration in the SN by TH immunohistochemistry. As showed in Figure 3B, in the control vehicle groups, dopaminergic neurons in the SN were intensely immunoreactive to TH (TH-ir) and there was no significant difference in the number of TH-ir neurons. The number of TH-ir neurons in the unlesioned side was not significantly different between groups (data not shown here). However, compared with the control and vehicle groups, the number of TH-ir neurons in the lesioned side of 6‑OHDA-treated rats was significantly reduced. Compared with the 6-OHDA group, the number of TH-ir neurons in the 6-OHDA plus high-dose ECH group was significantly higher (Fig. 3C, n = 5, P < 0.05).

ECH rescues protein and mRNA expression levels of TH and DAT, and decreases α-synuclein accumulation in 6-OHDA-treated rats

To determine the neurotoxicity of 6-OHDA and protective effect of ECH, we studied the protein and mRNA expression levels of TH, DAT and α-synuclein in the striatum of rats. As shown in Figure 4, treatment with 6-OHDA resulted in increased protein and mRNA expression levels of α-synuclein, indicating its accumulation. Decreased protein and mRNA expression levels of TH and DAT indicated the apoptosis of dopaminergic neurons. Compared with the 6-OHDA group, the 6-OHDA + ECH groups had significantly increased TH and DAT mRNA and protein expression, and reduced α-synuclein accumulation (n = 3, P < 0.05).

ECH protects dopaminergic neurons from 6-OHDA-induced TH down-regulation and seipin overexpression in vivo and inhibits ERS-associated protein Bip expression through seipin in vitro.

Effect of ECH on co-localization of TH and seipin in 6-OHDA-treated rats

As the neurotoxicity of 6-OHDA and protective effect of ECH in striatum were verified, we wondered if the protective effect occurring in the SNpc was associated with the reduction of seipin accumulation. To validate this hypothesis, tissue sections from the 6-OHDA group (Fig. 5A, model) and 6-OHDA + ECH (7 mg/kg) group (Fig. 5B, ECH) were stained for TH (red) and seipin (green), and the nuclei were counterstained with DAPI (blue). The sections were examined with a Zeiss Axiophot microscope equipped for epifluorescence illumination. Different channels were merged to obtain co-localization of seipin with the DA neuromarker, TH. As shown in Figure 5A, in the same rat's brain, after three-dimensional stereotactic injection of 6-OHDA, the number of TH-positive neurons in the SNpc was reduced and seipin expression increased on the lesioned side. In the ECH group, TH-positive neurons on the lesioned side were rescued from 6-OHDA neurotoxicity and the accumulation of seipin induced by 6-OHDA was decreased (Fig. 5B).

ECH inhibited ERS-associated protein Bip expression through seipin in vitro

According to the results in Figure 2, we verified that 6-OHDA could induce ER damage and ECH could attenuate this effect in PC12 cells. As shown in Figure 5A, 6-OHDA treatment caused damage to dopaminergic neurons and seipin overaccumulation. Therefore, we speculated that seipin overexpression might be related to ERS and could be regulated by ECH. In Figure 5C, the results demonstrated that seipin and the ERS-associated protein, Bip, were up-regulated by 6-OHDA administration, and that this accumulation was attenuated by ECH treatment. In Figure 5D, Bip accumulation induced by 6-OHDA was reversed by administration of the siRNA for seipin. These results indicated that ECH could decrease seipin overexpression induced by 6-OHDA and that seipin accumulation was a crucial factor leading to ERS.

ECH protects dopaminergic neurons from 6-OHDA-induced ERS-associated protein up regulation and inhibits seipin over accumulation in vivo

To further confirm the role of ECH in the ERS pathway and regulation of seipin expression in vivo, we studied protein and mRNA expression levels of seipin, GRP94, GRP78/BIP, ATF4 and CHOP in the striatum of rats. As shown in Figure 6, treatment with 6-OHDA resulted in increased protein (n = 5, rats’ striata pooled to reach a sufficient protein concentration, P < 0.05, Fig. 6A and B) and mRNA (n = 6) expression levels of GRP94, BIP, ATF4 and CHOP (Fig. 6C). Interestingly, the mRNA level of seipin was not significantly increased following 6-OHDA or ECH treatment, while the protein expression level of seipin could be affected by 6-OHDA and ECH. Therefore, we speculated that the accumulation or degradation of seipin was due to post-transcriptional regulation. 6-OHDA is known to activate various ERS markers and chaperones, and initiate their downstream cascades leading to an increase in CHOP, activating apoptosis. Compared with the 6-OHDA group, the 6-OHDA + ECH groups had significantly decreased expression of seipin, GRP94, BIP, ATF4 and CHOP (P < 0.05 or P < 0.01), which may explain how ECH protects dopaminergic neurons.

ECH protects against 6-OHDA-induced ERS through promoting seipin ubiquitination and degradation

To validate the hypothesis of ECH regulation of the ERS pathway through seipin, we knocked down seipin in PC12 cells using siRNA and measured ERS-associated protein expression. As shown in Figure 7A, the protein expression levels of seipin, GRP94, BIP, ATF-4 and CHOP were up-regulated by 6-OHDA and reversed by ECH. When seipin expression level was knocked down using siRNA, up-regulation of proteins in the ERS pathway induced by 6-OHDA was attenuated and further dramatically blocked by ECH (Fig. 7B). Furthermore, the results in Figure 7C indicated that the mRNA expression level of seipin was inhibited by siRNA but was not influenced by ECH or 6-OHDA. To determine whether ECH played a crucial role in seipin post-transcriptional regulation, seipin degradation was analysed in PC12 cells under MG132 treatment. As shown in Figure 7D, under MG132 treatment, 6-OHDA inhibited the degradation of seipin; however, ECH could accelerate this process. Therefore, we speculated the rescue degradation of seipin was relayed on ubiquitin-activating enzyme. More evidence of ECH promoting the ubiquitination of seipin was shown in Figure 7E; 6-OHDA interrupted the combination of ubiquitin and seipin, while ECH promoted the formation of seipin-ubiquitin compounds. These results demonstrate that ECH protects against 6-OHDA-induced ERS through promoting seipin ubiquitination and degradation.